Enzymatic synthesis of bile acid derivatives and biological evaluation against Trypanosoma cruzi

Article abstract of DOI:10.1016/j.bmc.2015.05.035

Enzyme catalysis was applied to synthesize derivatives of three bile acids and their biological activity was evaluated as growth inhibitors of the protozoan Trypanosoma cruzi. Twelve mono-, diacetyl and ester derivatives of deoxycholic, chenodeoxycholic and lithocholic acid, seven of them new compounds, were obtained through lipase-catalyzed acetylation, esterification and alcoholysis reactions in very good to excellent yield and a highly regioselective way. Among them, acetylated ester products, in which the lipase catalyzed both reactions in one-pot, were obtained. The influence of various reaction parameters in the enzymatic reactions, such as enzyme source, acylating agent/substrate ratio, enzyme/substrate ratio, solvent and temperature, was studied. Some of the evaluated compounds showed a remarkable activity as Trypanosoma cruzi growth inhibitors, obtaining the best results with ethyl chenodeoxycholate 3-acetate and chenodeoxycholic acid 3,7-diacetate, which showed IC₅₀: 8.6 and 22.8 μM, respectively. In addition, in order to shed light to bile acids behavior in enzymatic reactions, molecular modeling was applied to some derivatives. The advantages showed by the enzymatic methodology, such as mild reaction conditions and low environmental impact, make the biocatalysis a convenient way to synthesize these bile acid derivatives with application as potential antiparasitic agents.

Full text of DOI:10.1016/j.bmc.2015.05.035

Accepted Manuscript
Enzymatic synthesis of bile acid derivatives and biological evaluation against
Trypanosoma cruzi
Guadalupe García Liñares, M. Antonela Zígolo, Leandro Simonetti, Silvia A.
Longhi, Alicia Baldessari
PII:
S0968-0896(15)00445-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.05.035
BMC 12331
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
29 March 2015
13 May 2015
21 May 2015
Please cite this article as: García Liñares, G., Antonela Zígolo, M., Simonetti, L., Longhi, S.A., Baldessari, A.,
Enzymatic synthesis of bile acid derivatives and biological evaluation against Trypanosoma cruzi, Bioorganic &
Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.05.035
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Enzymatic synthesis of bile acid derivatives and biological evaluation
against Trypanosoma cruzi
a
a
b
b
Guadalupe García Liñares , M. Antonela Zígolo , Leandro Simonetti , Silvia A. Longhi
a
and Alicia Baldessari ∗
a
Laboratorio de Biocatálisis. Departamento de Química Orgánica y UMYMFOR, Facultad de Ciencias Exactas y Naturales,Universidad
de Buenos Aires, Ciudad Universitaria,Pabellón 2, piso 3, C1428EGA Buenos Aires, Argentina.
b
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular “Dr. Héctor N. Torres”, CONICET,
Vuelta de Obligado 2490, C1428ADN Buenos Aires, Argentina
Abstract
Enzyme catalysis was applied to synthesize derivatives of three bile acids and their
biological activity was evaluated as growth inhibitors of the protozoan Trypanosoma cruzi.
Twelve mono-, diacetyl and ester derivatives of deoxycholic, chenodeoxycholic and
lithocholic acid, seven of them new compounds, were obtained through lipase-catalyzed
acetylation, esterification and alcoholysis reactions in very good to excellent yield and a
highly regioselective way. Among them, acetylated ester products, in which the lipase
catalyzed both reactions in one-pot, were obtained. The influence of various reaction
parameters in the enzymatic reactions, such as enzyme source, acylating agent/substrate
ratio, enzyme/substrate ratio, solvent and temperature, was studied. Some of the evaluated
compounds showed a remarkable activity as Trypanosoma cruzi growth inhibitors,
obtaining the best results with ethyl chenodeoxycholate 3-acetate and chenodeoxycholic
acid 3,7-diacetate, which showed IC50: 8.6 µM and 22.8 µM respectively. In addition, in
order to shed light to bile acids behavior in enzymatic reactions, molecular modeling was
applied to some derivatives. The advantages showed by the enzymatic methodology, such
as mild reaction conditions and low environmental impact, make the biocatalysis a
convenient way to synthesize these bile acid derivatives with application as potential
antiparasitic agents.
Keywords: Bile acids; lipase-catalyzed; molecular modeling; Chagas disease
1
. Introduction
∗Corresponding author. Tel.: 5411-4576-3300 ext. 262; fax: 5411-4576-3385; e-mail: alib@qo.fcen.uba.ar

Bile acids are widely distributed in nature as oxygenated metabolites of steroids. The most
common bile acids are: chenodeoxycholic acid (CDCA, 1a), deoxycholic acid (DCA, 1b)
1
lithocholic (LCA, 1c) and cholic acid (CA, 1d) (Figure 1).
Figure 1. Structure of bile acids.
Bile acids are the end products of cholesterol catabolism in mammals and play a critical
role in biological processes such as digestion, secretion and the regulation of cholesterol
metabolism. They are of great interest by acting as powerful biological detergents that help
2
in dissolving and absorption of fats, fatty acids and lipid soluble vitamins and because they
exhibit various pharmacological activities, such as potent and selective farnesoid X receptor
3
4
5
(
FXR) antagonism, antimicrobial and anticancer activities.
Moreover, all bile acids and their conjugates form bile salts which, as amphiphilic
6
compounds, are widely used in drug formulations as absorption promoters and as
excipients that can influence gastrointestinal solubility, absorption and chemical or
7
8
enzymatic stability of drugs, enhance transdermal penetration, etc.
The application of enzymes and whole cells of microorganisms in the synthesis of
9
,10
pharmaceuticals derivatives has received considerable attention in recent years. It is well
recognized that enzymes are capable of accepting a wide array of substrates, and catalyze
several reactions in a regio-, chemo- and enantioselective way. As a result, biocatalysts
allow carrying out different chemical transformations without the need for tedious
protection and deprotection steps, especially in compounds with several functional
11
groups . Particularly, the application of lipases in non-aqueous media has been widely
used for several synthetic reactions such as esterification, transesterification, aminolysis,
1
2-14
polymerization, etc.
leading to the synthesis of pharmaceuticals and biologically active
1
5
compounds. Several studies on the esterification and transesterification of a variety of
substrates have shown that lipases are also useful in the steroid field, playing an important
role in the mild and selective interconversion of functional groups via regio- and
stereoselective transformation.
we have modified pregnanes and androstanes, synthesized fatty acid derivatives of
1
6,17
Taking into account these properties, in our laboratory
1
8
dehydroepiandrosterone, hydrocortisone and 3,17-β-estradiol, a series of novel 20-
1
9
succinates of pregnanes and acetyl cortexolone.

In the present paper, we want to report the results obtained through lipase-catalyzed
esterification, acylation and alcoholysis reactions in the preparation of derivatives of three
bile acids: chenodeoxycholic acid (CDCA, 1a), deoxycholic acid (DCA, 2a) and lithocholic
acid (LCA, 3a).
Although the pharmacological activity of bile acid derivatives has been extensively studied,
there is no evaluation of their biological activity as antiparasite so far. American
trypanosomiasis (Chagas’ disease) is among the most prevalent parasitic diseases
2
0
worldwide. It has been estimated that around 18–20 million people are infected and over
0 million individuals are at risk of infection by the hemoflagellated protozoan
4
2
1
Trypanosoma cruzi, the responsible agent of this disease. As other kinetoplastid parasites,
T. cruzi has a complex life cycle possessing four main morphological forms.
Transmission via the placenta or by blood transfusion is the responsible mechanism for the
occurrence of Chagas’disease in developed countries where it is not endemic. The
existing chemotherapy for Chagas’ disease is still deficient and mainly based on old and
empirically discovered drugs, often showing unwanted side effects. Therefore, the
development of a safe and effective chemotherapy involving new antiparasitic agents, is
essential.
Recently, it was established that the sterol biosynthesis pathway is an important metabolic
2
2
2
3
2
4
2
5
pathway in members of the Trypanosomatidae family. Also in the steroid field, some
examples of hybrids based on a steroidal framework have been reported as potential
antiparasitic agents.
2
6
Considering the role of steroids in trypanosome metabolism, our research in the field of
steroids and antiparasitic activity of organic compounds, we are also presenting the
evaluation of the biological activity of the bile acid derivatives synthesized in this work, as
growth inhibitors of the protozoan Trypanosoma cruzi.
2
. Results and Discussions
Bile acids exhibit a hydrophobic side (β) containing angular methyl groups and a
hydrophilic face (α) with hydroxyl groups. This structure is responsible for their
amphipathic nature and, consequently, in water bile acids self-associate to form multimers
above a critical concentration. Hydrogen bonding plays a key role in these multimers.
The ratio of the hydrophobic to the hydrophilic surface area of bile acids determines their
broad biological activity. Sometimes, their potential use is difficult because of their low
solubility in both hydrophilic and hydrophobic media. In order to find an increase in
lipophilicity and enhance permeability features, we planned to modify the structure of some
bile acids. For example, the presence of an acyl group at the C-3, C-7 or C-12 positions or
esterification of the acid moiety could make them more lipophilic. Taking this into account,
in the following section we are going to describe the results obtained by lipase-catalyzed
esterification, acetylation and alcoholysis reactions of CDCA, DCA and LCA.

2
.1. Enzymatic Esterification
According to literature, bile acid esters have been obtained by various procedures. For
example, chenodeoxycholic methyl ester was prepared using methanol and p-toluensulfonic
2
7
28
acid or methanesulfonic acid and also applying these conditions under microwave
2
9
irradiation. CDCA ethyl ester was described as an intermediate in the synthesis of
chenodiol by reduction of (5β,7α)-7-hydroxy-3-oxocholan-24-oic acid ethyl ester.
Triethylamine and EEDQ were used as catalysts in the synthesis of ethyl ester of
deoxycholic acid, and ethyl ester of lithocholic acid was biosynthesized by rat intestinal
microflora and its identity was confirmed by combined gas-liquid chromatography-
chemical ionization mass spectrometry.
3
0
3
1
3
2
With the aim to apply enzymes as an alternative procedure, we carried out the esterification
of CDCA, DCA and LCA with ethanol and lipases as catalysts (Scheme 1).
Scheme 1. Enzymatic synthesis of ethyl esters of bile acids CDCA (1a), DCA (1b)
and LCA (1c).
We studied the behavior of various lipases and some reaction parameters such as solvent,
temperature, enzyme:substrate ratio (E/S) and nucleophile (ethanol): substrate ratio (Nu/S)
to achieve the optimal conditions. In every case CDCA (1a) was used as bile acid model.
2
.1.1. Enzyme screening and solvent effect
Three commercial lipases were evaluated in the esterification reaction of 1a with ethanol:
Candida rugosa lipase (CRL), Candida antarctica lipase B (CAL B) and Lipozyme, lipase
from the fungus Rhizomucor miehei (LIP). The solvents tested were acetonitrile, hexane,
diisopropyl ether and toluene and the reaction without co-solvent using ethanol both as
nucleophile and solvent. Reactions were carried out at 55 °C using an enzyme:substrate
ratio E/S: 10, ethanol:substrate ratio: 5 and the time necessary to achieve maximum
conversion (Table 1). In the absence of biocatalyst no product was obtained and it was
observed that enzyme activity was variable, giving CAL B the most satisfactory results in
hexane: product 2a at 100% conversion in 24 h of reaction (Table 1, entry 2). This lipase

was also active in toluene and DIPE, but to a lesser extent (Table 1, entries 3 and 4). In
hexane, LIP was also active but it showed a lower performance than CAL B showing 70%
conversion after 96 h, whereas with CRL the enzyme activity was very low. Working
without co-solvent, in neat ethanol, CAL B also afforded the desired product with 100 %
conversion but at a longer reaction time (72 h) (Table 1, entry 1).
Table 1. Optimization of reaction parameters for lipase-catalyzed esterification of CDCA.
T (°C) E/S[a]
Entry
Lipase
Solvent
Nu/S
t (h)
Conversion
Lipase and solvent
1
2
3
4
5
6
7
8
9
CAL B
CAL B
CAL B
CAL B
CAL B
LIP
Ethanol
Hexane
DIPE
55
55
55
55
55
55
55
55
55
55
55
10
10
10
10
10
10
10
10
10
10
10
5
5
5
5
5
5
5
5
5
5
5
72
24
72
72
96
96
96
96
96
96
96
100
100
40
60
15
60
70
25
30
15
20
Toluene
MeCN
Ethanol
Hexane
DIPE
LIP
LIP
LIP
Toluene
Ethanol
Hexane
1
0
1
CRL
1
CRL
E/S
1
1
1
1
2
CAL B
CAL B
CAL B
CAL B
Hexane
Hexane
Hexane
Hexane
55
55
55
55
15
5
5
5
5
5
24
24
24
24
100
70
3
4
5
2
40
1
35
Nu/S
1
1
1
6
7
8
CAL B
CAL B
CAL B
Hexane
Hexane
Hexane
55
55
55
10
10
10
10
2
24
24
24
100
72
1
60
Temperature
1
9
CAL B
Hexane
Hexane
25
35
10
10
5
5
24
24
30
56
2
0
CAL B
a
Alcohol: ethanol. E/S: enzyme amount in mg/substrate amount in mg.

2
.1.2. Effect of enzyme:substrate ratio
The influence of the enzyme:substrate ratio in the enzymatic esterification was evaluated at
4 h, using ethanol as nucleophile at Nu/S ratio of 5, hexane as co-solvent at 55 °C and
2
variable amounts of CAL B. From the results (Table 1, entries 12-15), it can be concluded
that a ratio E/S of 10 (Table 1, entry 2) is the most satisfactory.
2
.1.3. Effect of nucleophile:substrate ratio
The influence of the Nu/S ratio on esterification yield was evaluated at 24 h in hexane using
CAL B (E/S = 10) at 55 °C and variable amounts of nucleophile. From the results (Table 1,
entries 16-18), it can be concluded that a ratio E/S of 5 is the most satisfactory.
2
.1.4. Influence of temperature
Investigating the influence of temperature on the enzymatic esterification, we performed
the reaction at 25 °C, 35 °C and 55 ºC. The other reaction parameters were settled to their
optimal values (CAL B, hexane, E/S: 10 and Nu/S: 5). The results (Table 2, entries 2, 19
and 20) show an increase in yield with the increase in temperature. Therefore we selected
5
5 °C as the reaction temperature.
Considering the previously mentioned experiments, the following standard conditions for
the enzymatic esterification of chenodeoxycholic acid were chosen: CAL B as biocatalyst,
temperature: 55 °C, E/S: 10, Nu/S: 5 and hexane as solvent.
2
.1.5. Lipase-catalyzed esterification of DCA and LCA
Using the optimal conditions described for chenodeoxycholic acid in the previous section,
we applied the enzymatic strategy in the esterification of deoxycholic (1b) and lithocholic
(
1c) acids. All ethyl esters were obtained in excellent yield (Ethyl CDCA: 99%, Ethyl
DCA: 89% and Ethyl LCA: 93%), although the reaction time was higher for obtaining 2b
and 2c (72 h).
2
.2. Enzymatic acetylation
At our best knowledge, the preparation of monoacetyl derivatives of CDCA, DCA and
LCA from the corresponding free acids have not been reported, all related data involve the
methyl esters as starting materials. For example, chenodeoxycholic acid methyl ester was
acetylated using acetic anhydride, pyridine and DMAP at room temperature during eight
hours to lead the methyl ester-7-acetate derivative, whereas without DMAP in sixteen hours
3
3
the methyl ester-3-acetate derivative was obtained.
The peracetylated methyl chenodeoxycholate 3,7-diacetate was prepared from the
tosylhydrazone of its methyl ester in the presence of acetic acid and sodium borohydride at
3
4
6
0 °C. Regarding DCA, the reaction with ethyl acetate in p-toluensulfonic acid and water
3
5
at reflux afforded ethyl-DCA-3-acetate in 75% yield.

The presence of the two hydroxyl groups in CDCA makes this compound an interesting
model for studying the selectivity of the enzymatic acetylation. Therefore, at the beginning
we study the behavior of lipases in the acetylation of this bile acid. Then, we applied the
optimal reaction conditions to the other two bile acids and their corresponding ethyl esters
(
Scheme 2).
O
O
R2
R2
OR3
OR3
CH CO Et / lipase
3
2
R1
HO
R1
3
H CCO
2
1
2
3
1
1
1
2
2
2
a: R = OH, R = H, R = H
1
2
3
3
3
4
4
4
a: R = OH, R = H, R = H
1
2
3
b: R = H, R = OH, R = H
1
2
3
b: R = H, R = OH, R = H
1
2
3
c: R = H, R = H, R = H
1
2
3
a: R = OH, R = H, R = CH CH
2
3
1
2
3
a: R = OH, R = H, R = CH CH
1
2
3
2
3
b: R = H, R = OH, R = CH CH
2
3
1
2
3
b: R = H, R = OH, R = CH CH
1
2
3
2
3
c: R = H, R = H, R = CH CH
2
3
1
2
3
c: R = H, R = H, R = CH CH
2
3
Scheme 2. Enzymatic synthesis of acetylated derivatives of bile acids CDCA (1a), DCA
1b) and LCA (1c).
(
2
.2.1. Enzyme screening and solvent effect
Again three commercial lipases were tested in the acetylation reaction of CDCA using ethyl
acetate as acylating agent: Candida rugosa lipase (CRL), Candida antarctica lipase B
(
CAL B) and Lipozyme (LIP). The solvents tested were acetonitrile, hexane, diisopropyl
ether and toluene and the reaction without co-solvent using ethyl acetate both as
nucleophile and solvent. Reactions were carried out at 55 °C using an enzyme:substrate
ratio E/S: 10, nucleophile:substrate ratio: 5 and the time necessary to achieve maximum
conversion (Table 2). In the absence of biocatalyst no product was obtained and it was
observed that enzyme activity was variable, giving CAL B the most satisfactory results in
hexane: product chenodeoxycholic acid 3-acetate (3a) at 100% conversion in 48 h of
reaction (Table 2, entry 2). This lipase was also active in toluene and DIPE, but to a lesser
extent (Table 2, entries 3-5). CRL also showed some activity but lower than CAL B (Table
2
, entries 10 and 11). Working without co-solvent with ethyl acetate as nucleophile and
solvent, CAL B also afforded the desired product with 100% conversion, but at 120 h of
reaction (Table 2, entry 1).
The enzymatic reaction allowed us to obtain the CDCA-3 acetate derivative in quantitative
yield and under simple and mild reaction conditions. In this case, the lipase behavior was
highly regioselective since the hydroxyl moiety on carbon 7 did not react at all under the
mentioned conditions. Since the reaction with ethyl acetate only afforded the monoacylated
product in the position 3, we decided to try the enzymatic acetylation using vinyl acetate
and isopropenyl acetate as acylating agents, well-known as active reagents in lipase-

catalyzed acetylations. In this case, the same product, 3a, was also obtained, but at a shorter
reaction time (24 h).
Table 2. Optimization of reaction parameters for lipase-catalyzed acetylation of CDCA.
E/Sa
Nu/Sb
Entry
Lipase
Solvent
T
°C)
t (h) Conversion
(
Lipase and solvent
1
2
3
4
5
6
7
8
9
CAL B
CAL B
CAL B
CAL B
CAL B
LIP
Ethyl acetate
Hexane
55
55
55
55
55
55
55
55
55
55
55
10
10
10
10
10
10
10
10
10
10
10
-
120
100
100
40
5
5
5
5
-
48
96
DIPE
Toluene
MeCN
96
60
120
96
15
b
Ethyl acetate
Hexane
80
b
LIP
5
5
5
-
96
53
b
LIP
DIPE
120
120
120
120
25
b
LIP
Toluene
Ethyl acetate
Hexane
30
1
0
1
CRL
40
50
1
CRL
5
E/S
1
1
1
2
3
4
CAL B
CAL B
CAL B
Hexane
Hexane
Hexane
55
55
55
5
2
1
5
5
5
48
96
95
90
85
120
Nu/S
1
1
1
5
6
7
CAL B
CAL B
CAL B
Hexane
Hexane
Hexane
55
55
55
5
5
5
10
2
48
48
48
90
80
65
1
Temperature
1
8
CAL B
CAL B
Hexane
Hexane
25
35
5
5
5
5
48
48
51
76
1
9
_
___________________________________________________________________________
a
b
E/S: enzyme amount in mg/substrate amount in mg. Entries 1, 6 and 10: Ethyl acetate:
c
nucleophile and solvent. Product: Ethyl chenodeoxycholate

It is important to note that LIP, with ethyl acetate without any other solvent, exhibited a
good performance, showing 80% conversion after 96 h, but surprisingly, the product
obtained was the ethyl ester (2a) and not the acetylated derivative 3a (Table 2, entries 6-9).
This interesting result suggests that, in the presence of LIP, esterification of the carboxyl
group was preferred even with the small amount of ethanol from the hydrolysis of the
acylating agent. As it is well-known, in enzymatic activity the solvent influence is
1
2
remarkable. Although LIP did not show to be the best biocatalyst in the esterification
reaction of CDCA (Table 1), using ethyl acetate as solvent afforded good results.
2
.2.2. Effect of enzyme:substrate ratio
The influence of E/S in the enzymatic acetylation was evaluated using Nu/S: 5, hexane as
solvent at 55 °C and variable amounts of CAL B. From the obtained results, it was
observed that E/S:10 is the best (Table 2, entry 2). However, as working at E/S:5,
conversion is slightly lower but considering that it is preferable to use the lowest amount of
enzyme (Table 2, entry 12), E/S: 5 was the ratio of choice.
2
.2.3. Influence of temperature
Temperatures of 25 °C, 35 °C and 55 °C were tested, keeping the reaction parameters at
their optimal values (CAL B, hexane, Nu/S:5, E/S: 5). Conversions of 51% at 25 °C, 76 %
at 35 °C and 100% at 55 °C were observed, showing the influence of temperature in yield
for the catalyzed reactions (Table 2, entries 12, 18 and 19). So we decided to perform the
reaction at 55 °C.
Taking into account these studies, we have chosen as optimal conditions for the enzymatic
acetylation of the bile acids: CAL B as biocatalyst, ethyl acetate as acylation agent, hexane
as solvent, temperature: 55 °C, E/S: 5 and Nu/S: 5.
2
.2.4. Acetylation of DCA and LCA
Once the experimental conditions were optimized, we applied them to the acetylation of
bile acids DCA 1b and LCA 1c. The results are summarized in Table 3. As it can be
observed, the acetylation of DCA led to the monoacetylated derivative in C3 (3b),
regioselectively and at shorter time than 1a. By contrast, in the case of lithocholic acid 1c,
ethyl lithocholate 3-acetate (4c) was obtained as the only product. This unexpected result is
of great interest since both: acetylation and esterification reactions were performed in only
one step. With the aim to obtain exclusively the acetylation product from lithocholic acid
we tried to favor the enzymatic acetylation over the esterification. Then, the reaction was
carried out with activated acylating agents such as isopropenyl acetate and vinyl acetate and
CAL B as biocatalyst. Again, it was obtained 4c as the only product but in a shorter
reaction time (16 h).
With LIP it was not possible to obtain acetylation products from 1b or 1c, only the
corresponding ethyl esters 2b and 2c were obtained in very good yield.

Table 3. Enzymatic acetylation of bile acids and ethyl esters.
Bile acid
Lipase Acylating
agent
t
Product yield (%)
2
b
2c
3a
3b
3c
4a
4b
4c
CDCA
DCA
LCA
DCA
LCA
CAL B
CAL B
CAL B
LIP
EtOAct
EtOAct
EtOAct
EtOAct
EtOAct
EtOAct
EtOAct
EtOAct
VinOAc
i-PrOAc
48
24
24
96
24
48
96
96
16
16
90
99
100
91
LIP
93
Et-CDCA CAL B
97
Et-DCA
Et-LCA
LCA
CAL B
CAL B
CAL B
CAL B
90
85
98
97
LCA
Experimental conditions: Solvent: hexane; Temperature: 55 °C; E/S: 5:1; Nu/S: 5:1, t: time in hours. VinOAc:
vinyl acetate, i-PrOAc: isopropenyl acetate.
The standard conditions were also applied to acetylation of ethyl esters 2a-c. In all cases,
acetylation of hydroxyl group of 3-position was observed, obtaining the corresponding
ethyl esters 3-acetates 4a-c.
2
.3. Enzymatic alcoholysis
The good regioselectivity of CAL B in catalyzing the acetylation of 3 hydroxyl group of
the bile acids, prompted us to study if this lipase showed the same behavior in the
alcoholysis reaction on the peracetylated chenodeoxycholic acid (5a) and deoxycholic acid
(
5b) (Scheme 3).
The substrates for the enzymatic alcoholysis 5a and 5b, were obtained in 95% and 90%
yield respectively, by treatment of the corresponding acids 1a and 1b with acetic anhydride
and pyridine at room temperature during 16 h.
Scheme 3. Chemical synthesis and enzymatic alcoholysis of peracetylated derivatives of
CDCA and DCA.
At this point, we attempted to carry out the enzymatic alcoholysis of 5a-b using CAL B as
biocatalyst, ethanol, n-butanol and n-octanol as nucleophiles and acetonitrile, diisopropyl

ether and hexane as solvents. The results are presented in Table 4. The best results were
achieved with ethanol and hexane as solvent. Higher alcohols such as n-butanol and n-
octanol did not show good performance. Again the lipase showed a regioselective behavior
acting only at 3-position. Under these conditions, it was possible to obtain ethyl
chenodeoxycholate acid 7-acetate (7a) from 5a in quantitative yield, and deoxycholic acid
1
2-acetate (6b) from 5b in good yield (Table 4, entries 1, 2 and 5). The site of alcoholysis
in every case was unambiguously established by NMR spectroscopic analysis. These
results prove that in the alcoholysis the enzyme maintains the same regioselectivity than in
acetylation reactions, offering a good alternative to remove acyl groups on carbon 3 while
keeping unaltered the other positions. This fact is in agreement with previous work
1
5,18,19
performed in our laboratory using pyridoxine, terpene and steroids as substrates.
It is interesting to observe that the reaction with the diacetylated derivative of
chenodeoxycholic acid 5a afforded a 7-monoacetylated ethyl ester (7a), working ethanol as
alcoholysis and esterifying agent at the same time.
Table 4. CAL B-catalyzed alcoholysis of CDCA-3,7-diacetate (5a) and DCA-3,12-
diacetate (5b).
a
Entry Substrate
Solvent
Nu
Nu/S
t (h)
Conversion (%)
7a
6b
1
2
3
4
5
6
7
8
9
5a
5a
5a
5a
5a
5a
5a
5a
5b
5b
5b
5b
5b
5b
5b
5b
Hexane
Hexane
MeCN
MeCN
DIPE
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
BuOH
OctOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
BuOH
OctOH
1.2
5
120
48
100
100 (93)
n.d.
1.2
5
96
96
10
1.2
5
120
120
96
10
DIPE
30
Hexane
Hexane
Hexane
Hexane
MeCN
MeCN
DIPE
5
n.d.
5
120
120
120
120
48
n.d.
1.2
5
25
1
0
1
2
3
4
5
6
73(65)
n.d.
n.d.
10
1
1
1
1
1
1
1.2
5
1.2
5
96
DIPE
120
48
40
Hexane
Hexane
5
n.d.
n.d.
2
48
_
_________________________________________________________________________________
Experimental conditions: Enzyme: CAL B; Solvent: hexane; Temperature: 55 °C; E/S: 5:1; Nu/S: 5:1.
a
n.d.: non detected. Entries 2 and 10: % yield in brackets.

2
.4. Molecular Modeling
To shed light to the molecular determinants of the enzymatic reactions on bile acids, we
examined the enzymatic acetylation using molecular modeling. This approach helps to
understand the structural relation between the enzymatic pocket and the characteristic
steroidal framework, which accounts for the selectivity of the biological behavior in this
family of natural compounds. Then, molecular docking studies have been performed on
chenodeoxycholic acid, deoxycholic acid and lithocholic acid with CAL B and the ability
of the catalytic pocket to accommodate these substrates was evaluated. The analysis of the
results led to the selection of a possible conformation for each bile acid, using the
interaction with the amino acids of the catalytic site (Asp187-His224-Ser105), binding
energy and the population of the clusters as selection criteria. CDCA, DCA and LCA
interact with the active site (Ser105) of the enzyme through C3-OH showing distances of
1
.94 Å 1.95 Å and 1.95 Å and binding energies of 5.36 kcal/mol, 7.13 kcal/mol and 6.13
kcal/mol, respectively. The main favorable conformations correspond to those in which the
hydroxyl group on carbon 3 points toward the catalytic triad.
In the case of CDCA and DCA, the distances of the hydroxyl groups in position 7 and 12
respectively, located in the steroid B ring, were 4.38 Å and 4.73 Å. These hydroxyl groups
are farther from the catalytic serine than C3-OH justifying their lack of reactivity
a)
b)
Ser 105
His 224
Asp 187
c)
Asp 187
His 224
Ser 105
Figure 2. Docking of CAL B with: a) CDCA b) DCA and c) LCA

The docking representations of CAL B with CDCA, DCA and LCA are presented in Figure
. In every case it can be observed the hydrophobic tunnel through which the substrates
2
enter to reach the catalytic triad of the lipase.
We also used molecular modeling to explain the different products obtained by reaction of
bile acids with ethyl acetate in hexane catalyzed by CAL B.
Under the same reaction conditions CDCA and DCA afforded the 3-acetyl derivatives.
Instead LCA gave ethyl lithocholate 3-acetate, indicating that the lipase catalyzed both
reactions: acetylation and esterification.
In previous work involving a combination study of docking simulations and QSAR analysis
it was reported that in CAL B, among the explored five molecular interaction fields, the
3
6
contribution of the electrostatic field to the reactivity was the most significant. Taking this
into account, hydrogen bond interactions between polar residues in CAL B and the
hydroxyl groups in the steroid B ring in CDCA (C-7) and DCA (C-12), would result in a
higher stabilization of these substrates in the enzyme pocket than LCA. As a consequence
only the acetylation reaction was catalyzed with CDCA and DCA while ethyl-lithocholate
3
-acetate, through lipase- catalyzed acetylation and esterification, was obtained from LCA
under the same reaction conditions.
It could be assumed that acetylation could occur prior to esterification, leading to the
formation of lithocholic acid 3-acetate (3Aco-LCA) in the first step of the process. Then,
CAL B catalyzed the esterification of 3AcO-LCA with the ethanol released during the
acetylation with ethyl acetate.
In Figure 3, a docking study performed on 3AcO-LCA shows the entrance of the acetylated
substrate to the catalytic pocket by the end of the carboxylic acid group. It can be observed
a distance of 3.14 Å from carboxylic carbon to the catalytic serine and a binding energy of
6
.47 kcal/mol.
Asp 187
His 224
Ser 105
Figure 3. Docking of lithocholic acid 3-acetate

2
.5. Biological Evaluation
Lipids have essential roles in biological membranes and are sources of stored energy.
Recently, it was established that the sterol biosynthesis pathway is an important metabolic
2
5
pathway in members of the Trypanosomatidae family. Unlike mammals, which synthesize
cholesterol, epimastigote forms of T. cruzi produce mostly ergosterol. The sterol
biosynthesis pathway is therefore considered one of the most attractive targets for the
specific treatment of Chagas disease, and several enzymes from this pathway have been
3
7
studied as possible treatment targets.
Moreover, different azasterols have been evaluated as structural analogues of steroids in
trypanosomes. They function as inhibitors of key pathways in the synthesis of steroids,
having the enzyme sterol 24-methyltransferase as molecular target in Leishmania species
3
8
and Trypanosoma cruzi.
Taking into account that bile acids are considered as lipid metabolism regulators, the
reported bile acid derivatives could be inhibitors of T. cruzi sterol biosynthesis. Therefore,
all synthesized compounds were evaluated as growth inhibitors against the epimastigote
form of Trypanosoma cruzi. The assays were performed at concentrations ranging from 300
µM to 0.14 µM in three independent assays. Axenic parasites were incubated with different
concentrations of bile acid derivatives to obtain a dose–response curve that allowed
calculation of the IC50 values. Benznidazole (Bz), the only one drug used for Chagas’
treatment, was used as a positive control. Table 5 shows the results of only active
compounds. A remarkable inhibitory effect on T.cruzi growth was observed in the case of
the compounds ethyl chenodeoxycholate 3-acetate (4a) and chenodeoxycholic acid 3,7-
diacetate (5a), exhibiting IC50 values of 8.6 and 22.8 µM, respectively. It is interesting to
note that compound 4a resulted two fold more potent than benznidazole and compound 5a
showed a potency of the same order.
Table 5. IC50 values for Trypanosoma cruzi growth inhibition by bile acids derivatives
Compound
1a
2c
4a
5a
7a
Bz
IC50 (µM)
236.1 ± 3.1
266.3 ± 3.9
8.6 ± 0.2
22.8 ± 1.3
295 ± 5.2
18.8 ± 0.3
Figures 4 and 5 show the growth curve of epimastigote T. cruzi in the presence of
compounds 4a and 5a, respectively. Compounds 1a, 2c and 7a showed inhibition but at
lower extent and the rest of the tested compounds were inactive even at a concentration of
3
00 µΜ.

It can be observed that CDCA derivatives are the most active products and CDCA was the
only bile acid that showed activity. Particularly in this case, it seems that two key factors
improve the biological activity of the bile acid derivatives: the presence of an acetate group
in 3-position of ring A and the ethyl group esterifying the acid moiety in the side chain.
Both of them substantially increase the inhibitory activity displayed by CDCA, showing an
IC50 value of about 30 times lower. It is also interesting to note that the presence of only
one of these two groups has the opposite effect because both ethyl ester (2a) and 3-acetate
(
3a) did not exhibited antiparasitic activity. Furthermore, the presence of an acetate group
in the 7 position maintaining the ester function, did not improved the activity of CDCA (7a
vs 4a). In addition, two acetate groups at 3- and 7- positions also caused an increase in the
growth inhibition, being 5 to 10 times more potent than 1a. It could be assumed that the
presence of an acetate group in the 3-position is essential for activity. Moreover, it is also
required the presence of another group, acetate or ethyl ester that increases the lipophilicity
of the compound, which clearly improve the percent inhibition. The same applies for the
case of LCA, showing no activity, but with the ethyl ester showing a slight inhibition
although at a high concentration. In the case of DCA, none of the derivatives was active,
probably due to the presence of OH or AcO group in the position 12, which would not
allow an adequate interaction of the compound with the corresponding molecular target.
In summary, the observed results indicate that the presence of functional groups in
positions 3 and 7 of the steroid skeleton favors the antiparasitic activity of bile acid
derivatives. In particular, the presence of acetyl group in position 3 rather than position 7
increases the inhibitory activity exhibited by CDCA.
Figure 4. Effect of ethyl chenodeoxycholate 3-acetate (4a) concentration on
Trypanosoma cruzi growth.

Figure 5. Effect of chenodeoxycholic acid 3,7-diacetate (5a) concentration on
Trypanosoma cruzi growth.
3
. Conclusions
This work describes the application of enzymes to the synthesis of derivatives of the three
bile acids CDCA, DCA and LCA in a highly regioselective way. Scheme 4 shows the
performed reactions and the obtained products.
Among the tested enzymes, Candida antarctica B lipase gave the best results in
esterification, acylation and alcoholysis reactions. To begin, CDCA, DCA and LCA ethyl
esters were obtained as the only reaction products. The three esters were completely
characterized by spectroscopic methods and, in the case of Ethyl CDCA and Ethyl DCA,
obtained in higher yield than those reported by chemical synthesis (89% vs 78% and 93%
vs 19% respectively). Regarding Ethyl LCA, in previous work was not isolated and its
identity was only determined by GC-MS.
Lipase-catalyzed acetylation allowed us to obtain two new monoacetyl bile acid derivatives
in the position 3 of ring A in the steroid skeleton: 3-AcO-CDCA, 3-AcO-DCA. The
reaction was highly regioselective, neither 7-OH in CDCA nor 12-OH in DCA was
transformed. Under the same reaction conditions LCA afforded the acetyl derivative of the
ethyl ester by catalysis of CAL B for acetylation and esterification reactions in one pot.
The enzymatic acetylation of the ethyl esters also afforded the 3-monoacetylated products.
Now two new products were obtained, Ethyl 3-AcO-CDCA and Ethyl 3-AcO-LCA.
Finally, it is interesting to remark that the treatment of CDCA with Lipozyme under
acetylation conditions afforded the corresponding ethyl ester instead of the acetylation
product.

O
R3
OEt
CAL B
R1
R2
O
EtOH/hexane
H
Ethyl CDCA (2a), 24 h
Ethyl DCA (2b), 72 h
Ethyl LCA (2c), 72 h
OEt
HO
OAc
Ethyl 7-AcO-CDCA (7a)
CAL B
EtOAc/hexane
LIP
EtOAc/hexane
CAL B
EtOH/hexane
(only 2a)
O
R3
OEt
O
H
AcO
R2
OH
Ethyl 3-AcO-CDCA (4a), i) 48 h, ii) 16 h
Ethyl 3-AcO-DCA (4b), i) 96 h, i) 16 h
Ethyl 3-AcO-LCA (4c), i) 96 h, i) 16 h
O
R2
AcO
OAc
OH
only 4c
CAL B
Ac2O / Py
0 °C
3
,7-diAcO-CDCA (5a)
i) EtOAc/hexane
ii) VinOAc or i-PrOAc/
hexane
O
7
R3
R1
HO
OH
O
OAc
CDCA 1a: R1 = OH, R2 = H
DCA 1b: R1 = H, R2 = OH
OH
LCA 1c: R1 = H, R2 = H
AcO
R2
3
-AcO-CDCA (3a), 16 h
AcO
H
3-AcO-DCA (3b),16 h
3
,12-diAcO-DCA (5b)
CAL B EtOH/hexane
O
OAc
OH
HO
H
1
2-AcO-DCA (6b)
Scheme 4. Reactions and products from bile acids: CDCA, DCA and LCA.
By lipase-catalyzed alcoholysis of the peracetylated derivatives of CDCA and DCA it was
possible to obtain two new products: the complementary acetates 7-AcO in CDCA and 12-
AcO in DCA. Here, it is also interesting that in the case of CDCA, both alcoholysis of
acetyl group and esterification of the carboxyl group gave the acetylated ester Ethyl 7-AcO-
CDCA.

In summary, the enzymatic reactions offer a good alternative to synthesize ester and
monoacetylated derivatives from bile acids. Although the synthesis of these compounds
performed by chemical methods is not difficult, it has the disadvantage of using hazardous
reagents such as acetic anhydride, pyridine, and strong acidic media. The lipase-catalyzed
procedure uses ethanol as reagent for esterification and alcoholysis reactions and ethyl
acetate as acylating agent. The enzymatic approach also shows other interesting
advantages: the reaction is simple, it is performed at low temperature, and the products are
isolated by simple filtration and solvent evaporation. The lipase is biodegradable and,
consequently, more friendly to the environment than chemical catalysts. In addition,
because the enzyme is insoluble in the reaction medium, it is easily removed by filtration
and can be reused. In the esterification and acetylation of CDCA, CAL B retained 90 % and
8
0 % activity after five reaction cycles respectively.
Molecular modeling was applied to rationalize the selective behavior of CAL B and the
one- pot acetylation and esterification in the case of LCA.
All of the compounds were evaluated against epimastigote Trypanosoma cruzi. Compounds
4
a and 5a showed to be very potent as inhibitors of parasite growth with IC50 = 8.6 and
2.8 µM (benznidazole: 18.8 µM). Considering this data, these products can be useful as
2
potential drugs for chemotherapy of Chagas’ disease.
4
4
. Experimental
.1. General
Chemicals and solvents were purchased from Merck Argentina and Sigma-Aldrich de
Argentina and used without further purification. . Lipase from Candida rugosa (CRL) (905
U/mg solid) was purchased from Sigma Chemical Co.; Candida antarctica lipase B (CAL
B): Novozym 435 (7400 PLU/g) and Lipozyme RM 1M (LIP) (7800 U/g) were generous
gifts of Novozymes Spain; all enzymes were used "straight from the bottle". Enzymatic
reactions were carried out on Innova 4000 digital incubator shaker, New Brunswick
Scientific Co. at the corresponding temperature and 200 rpm. E/S: enzyme amount in
mg/substrate amount in mg. To monitor the reaction progress aliquots were withdrawn and
analyzed by TLC performed on commercial 0.2 mm aluminum-coated silica gel plates
(
F254), using EtOAc:hexane 3:7 as developing solvent and visualized by 254 nm UV or
immersion in an aqueous solution of (NH Mo O (0.04 M), Ce(SO (0.003 M) in
concentrated H SO (10%). % conversion was determined by gas chromatography on a
4
)
6
7
O24.4H
2
)
4 2
2
4
Thermo Focus GC chromatograph equipped with a flame ionization detector and a using
HP-17 column (10 m x 0.53 mm ID, 0.25 thickness; Agilent Corporation, USA). Nitrogen
was the carrier gas. Both injector and detector temperatures were set at 250 and 300 °C
respectively. Column temperature was programmed from 100 to 200 °C at a rate of
1
0 °C/min and stable at 200 °C for 1 min, then at a rate of 15 °C/min up to 290 °C and
stable at this temperature for 10 min. Melting points were measured in a Fisher Johns

apparatus and are uncorrected. Optical rotation values were measured in a CHCl solution
3
1
13
with a Perkin Elmer-343 automatic digital polarimeter at 25 °C. H NMR and C NMR
spectra were recorded at room temperature in CDCl as solvent using a Bruker AM-500
NMR instrument operating at 500.14 MHz and 125.76 MHz for H and C respectively.
3
1
13
1
The H NMR spectra are referenced with respect to the residual CHCl
solvent CDCl at δ = 7.26 ppm. Coupling constants are reported in Hertz (Hz). C NMR
spectra were fully decoupled and are referenced to the middle peak of the solvent CDCl at
3
proton of the
1
3
3
3
δ = 77.0 ppm. Splitting patterns are designated as: s, singlet; d, doublet; t, triplet; q,
quadruplet; qn, quintet; dd, double doublet, etc. IR spectra were recorded with a Nicolet
Magna 550 spectrometer. High Resolution Mass Spectrometry was recorded with Thermo
Scientific EM/DSQ II – DIP. The results were within ±0.02% of the theoretical values.
4
.2. Enzymatic esterification. General procedure
CAL B (1g) was added to a solution of the corresponding bile acid (100 mg) in hexane (10
mL) and ethanol (0.15 mL). The suspension was shaken (200 rpm) at 55 °C and the
progress of the reaction monitored by TLC/GC. Once the reaction was finished, the enzyme
was filtered off and the solvent evaporated under reduced pressure. The residue was
purified by column chromatography on silicagel employing mixtures hexane:ethyl acetate
as eluent (9:1-1:1). Reuse experiments: the filtered and washed enzyme was used in the
next enzymatic esterification under the same reaction conditions.
4
.3. Enzymatic acetylation. General procedure
CAL B (500 mg) was added to a solution of the corresponding bile acid (100 mg) in hexane
10 mL) and ethyl acetate (0.25 mL). The suspension was shaken (200 rpm) at 55°C and
(
the progress of the reaction monitored by TLC/GC. Once the reaction was finished, the
enzyme was filtered off and the solvent evaporated under reduced pressure. The residue
was purified by column chromatography on silicagel employing mixtures hexane:ethyl
acetate as eluent (9:1-1:1). Reuse experiments: the filtered and washed enzyme was used in
the next enzymatic acetylation under the same reaction conditions.
4
.4. Chemical peracetylation
In a typical procedure, a mixture of the corresponding bile acid (400 mg, 1 mmol) acetic
anhydride (0.5 mL, 5 mmol) and pyridine (1 mL) was stirred at room temperature. The
progress of the reaction was monitored by TLC. After completion of the reaction, the
mixture was partitioned between saturated solution ammonium chloride (10 mL) and
methylene chloride (10 mL). The aqueous phase was extracted with methylene chloride (3
x 10 mL). The combined organic layers were washed with saturated solution of sodium
chloride (5 x 10 mL), dried (MgSO4), and the solvent was evaporated. The residue was
purified by column chromatography (silica gel) employing mixtures of hexane/EtOAc as
eluent (9:1–7:3).

4
.5. Enzymatic alcoholysis. General procedure
To a solution of the peracetylated bile acid (100 mg) in 10 ml of the indicated solvent
containing 5 mol equiv of ethanol, CAL B (1 g) was added. The suspension was shaken
(
200 rpm) at 55 °C and the progress of the reaction was monitored by TLC. After indicated
time, the enzyme was filtered off, the solvent was evaporated, and the crude residue
purified by column chromatography on silica gel employing mixtures hexane:ethyl acetate
as eluent (9:1-1:1).
2
5
1
Ethyl Chenodeoxycholate (2a): Yield 99%; colorless oil; [α]
NMR (CDCl , 500 MHz) δ 4.12 (2H, q, J = 6.8 Hz, -OCH CH
Hz, H-7), 3.46 (1H, tt, J = 11.3, 4.2 Hz, H-3), 2.33 (1H, m, H-23b), 2.20 (1H, m, H-23a),
.25 (3H, t, J = 6.7 Hz, OCH CH ), 0.92 (3H, d, J = 6.4 Hz, H-21), 0.90 (3H, s, H-19), 0.65
3H, s, H-18); C NMR (CDCl , 500 MHz) d 174.4 (C-24), 72.0 (C-7), 68.5 (C-3), 60.2
OCH CH ), 31.0 (C-23), 22.8 (C-19), 18.3 (C-21), 14.2 (-OCH CH ), 11.8 (C-18). HRMS:
M + Na] Calcd. C26 443.3132.
D
+ 6.4° (c 1.0, CHCl
3
). H
3
2
3
), 3.84 (1H, dd, J = 2.5, 3.0
1
2
3
1
3
(
(
[
3
2
3
+
2
3
H
44NaO
4
443.3137. Found: C26
H44NaO
4
2
5
1
Ethyl Deoxycholate (2b): Yield 89%; colorless oil; [α]
NMR (CDCl , 500 MHz) δ 4.12 (2H, q, J = 6.7 Hz, -OCH
Hz, H-12), 3.60 (1H, tt, J = 11.0,4.0 Hz, H-3), 2.34 (1H, m, H-23b), 2.22 (1H, m, H-23a),
.24 (3H, t, J = 6.7 Hz, OCH CH ), 0.96 (3H, d, J = 6.5 Hz, H-21), 0.90 (3H, s, H-19), 0.67
3H, s, H-18); C NMR (CDCl , 500 MHz) d174.3 (C-24), 73.1 (C-12), 71.8 (C-3), 60.2
OCH CH ), 30.9 (C-23), 23.1 (C-19), 17.3 (C-21),14.2 (-OCH CH ), 12.7 (C-18). HRMS:
M + Na] Calcd. C26 443.3141.
D
+ 33.6 ° (c 1.0, CHCl
3
).
H
3
2
CH ), 3.97 (1H, dd, J = 2.5, 3.0
3
1
2
3
1
3
(
(
[
3
2
3
+
2
3
H
44NaO
4
443.3137. Found: C26
H44NaO
4
2
5
Ethyl Lithocholate (2c): Yield 93%; white solid, mp 87–88 °C; [α]
D
+ 6.4 ° (c 1.0,
), 3.61 (1H, tt, J =
1
CHCl
3
). H NMR (CDCl
3
,500 MHz) δ 4.11 (2H, q, J = 6.6 Hz, OCH
2
CH
3
1
1.2, 4.2 Hz, H-3), 2.31 (1H, m, H-23b), 2.19 (1H, m, H-23a), 1.25 (3H, t, J = 6.6 Hz, -
1
3
OCH
2
CH
3
), 0.91 (3H, d, J = 6.4 Hz, H-21), 0.89 (3H, s, H-19), 0.63 (3H, s, H-18); C
, 500 MHz) δ 174.4 (C-24), 71.8 (C-3), 60.2 (OCH CH ), 31.0 (C-23), 23.3.
C-19), 18.2 (C-21), 14.2 (OCH CH ), 12.0 (C-18). HRMS: [M + Na] Calcd. C26
27.3188. Found: C26 427.3191.
NMR (CDCl
3
2
3
+
(
4
2
3
H44NaO
3
H44NaO
3
2
5
Chenodeoxycholic acid 3-acetate (3a): Yield 90%; white solid, mp 108.5–109 °C; [α]
D
1
+
9.6 ° (c 1.0, CHCl
3
). H NMR (CDCl
3
, 500 MHz) δ 4.58 (1H, tt, J = 11.3,4.4 Hz, H-3),
3
.85 (1H, dd, J = 3.0, 2.5 Hz, H-7), 2.38 (1H, m, H-23b), 2.25 (1H, m, H-23a), 2.01 (1H, s,
1
3
COCH
3
), 0.93 (3H, d, J = 6.4 Hz, H-21), 0.91 (3H, s, H-19), 0.66 (3H, s, H-18); C NMR
, 500 MHz) δ 179.2 (C-24), 170.8 (COCH ), 74.4 (C-7), 68.5 (C-3), 30.8 (C-23),
2.7 (C-19), 21.5 (CH CO-),18.2 (C-21), 11.8 (C-18).HRMS: [M + Na] Calcd.
457.2930. Found: C26 457.2925.
(
CDCl
3
3
+
2
C
3
26
H42NaO
5
H42NaO
5

2
5
Deoxycholic acid 3-acetate (3b): Yield 99%; colorless oil; [α]
D
+ 41.6 ° (c 1.0, CHCl ).
3
1
H NMR (CDCl3, 500 MHz) δ 4.72 (1H, tt, J = 11.0 Hz, H-3), 4.00 (1H, dd, J = 2.5, 3.0
), 0.98 (3H, s, H-
9), 0.93 (3H, d,J = 6.4 Hz, H-21), 0.69 (3H, s, H-18); 13C NMR (CDCl3, 500 MHz) δ
74.4 (C-24), 170.8 (COCH ), 74.4 (C-12), 72.5 (C-3), 31.0 (C-23), 23.2 (C-19), 17.5 (C-
Hz, H-12),2.33 (1H, m, H-23b), 2.24 (1H, m, H-23a), 2.03 (3H,s, COCH
3
1
1
2
4
3
+
1), 12.9 (C-18). HRMS: [M + Na] Calcd. C26
57.2927.
H42NaO
5
457.2930. Found: C28
H46NaO
5
2
5
Ethyl Chenodeoxycholate 3-acetate (4a): Yield97 %; white solid, mp 90–90.5 °C; [α]
D
1
+
36.8 ° (c 1.0, CHCl
3
). H NMR (CDCl
3
, 500 MHz) δ 4.58 (1H, tt, J = 11.2,4.2 Hz, H-3),
), 3.85 (1H, dd, J = 2.5, 3.0 Hz, H-7), 2.33 (1H, m, H-
3b), 2.20 (1H, m, H-23a), 2.00 (1H, s, COCH ), 1.25 (3H, t, J = 6.7 Hz, -OCH CH ), 0.92
, 500
), 31.0 (C-
), 11.8 (C-18). HRMS: [M +
485.3238.
4
2
.12 (2H, q, J = 6.7 Hz, OCH
2
CH
3
3
2
3
1
3
(
3H, d, J = 6.4 Hz, H-21), 0.91 (3H, s, H-19), 0.66 (3H, s, H-18); C NMR (CDCl
MHz) δ 174.3 (C-24), 170.8 (COCH ), 74.4 (C-7), 68.5 (C-3), 60.2 (OCH CH
3), 22.7 (C-19), 21.5 (CH CO-), 18.3 (C-21), 14.3 (OCH CH
485.3243. Found: C26
3
3
2
3
2
3
2
3
+
Na] Calcd. C28
H46NaO
5
H
44NaO
4
2
5
Ethyl Deoxycholate 3-acetate (4b): Yield 89%; colorless oil; [α]
D
+ 41.6 ° (c 1.0,
1
CHCl
3
). H NMR (CDCl3, 500 MHz) δ 4.71 (1H, tt, J = 11.3,4.5 Hz, H-3), 4.12 (2H, q, J =
6
.5 Hz, OCH
H-23a), 2.02 (3H,s, COCH
H-21), 0.92 (3H, s, H-19),0.68 (3H, s, H-18); 13C NMR (CDCl3, 500 MHz) δ174.3 (C-24),
70.7 (COCH ), 74.3 (C-12), 73.2 (C-3),60.2 (OCH CH ),30.9 (C-23), 23.1 (C-19), 21.5
CH CO-),
Calcd.C28
2
CH
3
), 3.98 (1H, dd, J = 2.5, 3.0 Hz, H-12),2.33 (1H, m, H-23b), 2.23 (1H, m,
3
), 1.25 (3H, t, J = 6.7 Hz, -OCH CH ),0.97 (3H, d, J = 6.5 Hz,
2
3
1
(
3
2
3
+
3
17.4 (C-21), 14.3 (OCH
485.3243. Found: C26
2
CH
3
),12.7 (C-18). HRMS: [M + Na]
485.3240.
H
46NaO
5
H44NaO
4
2
5
Ethyl Lithocholate 3-acetate (4c): Yield 75%; white solid, mp 151–151.5 °C; [α]
D
+
1
2
4.4 ° (c 1.0, CHCl
3
). H NMR (CDCl
3
, 500 MHz) δ 4.70 (1H, tt, J = 11.2, 4.2 Hz, H-3) ,
), 2.32 (1H, m, H-23b), 2.19 (1H, m, H-23a), 2.02 (1H, s,
), 1.24 (3H, t, J = 6.7 Hz, -OCH CH ), 0.91 (3H, d, J = 6.4 Hz, H-21), 0.89 (3H, s,
H-19), 0.63 (3H, s, H-18); C NMR (CDCl , 500 MHz) δ 174.3 (C-24), 170.7 (COCH ),
4.4 (C-3), 60.2 (OCH CH ), 31.0 (C-23), 23.3 (C-19), 21.4 (CH CO-), 18.3 (C-21), 14.2
OCH CH ), 12.0 (C-18). HRMS: [M + Na] Calcd.C28
469.3289.
4
.10 (2H, q, J = 6.7 Hz, OCH
2
CH
3
COCH
3
2
3
1
3
3
3
7
(
2
3
3
+
2
3
H
46NaO
4
469.3294. Found:
C
28
H46NaO
4
3
9
Chenodeoxycholic acid 3,7-di-acetate (5a): Yield 90%; white solid, mp 96–97°C (lit.
2
5
1
mp 99 °C (dec.)); [α]
dd, J = 3.0, 2.5 Hz, H-7), 4.58 (1H, tt, J = 11.2, 4.3 Hz, H-3) , 2.38 (1H, m, H-23b), 2.27
1H, m, H-23a), 2.03 (3H, s, COCH ), 2.05 (3H, s, COCH ), 0.93 (3H, d, J = 6.4 Hz, H-21),
.93 (3H, s, H-19), 0.65 (3H, s, H-18); C NMR (CDCl , 500 MHz) δ 179.2 (C-24), 170.7
D
+ 8.5 ° (c 1.0, CHCl
3
). H NMR (CDCl
3
, 500 MHz) δ 4.88 (1H,
(
3
3
1
3
0
3

(
)
4
COCH
, 21.5 (CH
99.3036. Found: C28
3
), 170.5 (COCH
CO-), 18.2 (C-19), 11.7 (C-18). HRMS: [M + Na] Calcd.C28
499.3032.
3
), 74.2 (C-7), 71.3 (C-3), 30.8 (C-23), 22.7 (C-21), 21.6 (CH CO-
3
+
3
H44NaO
6
H
44NaO
6
4
0
Deoxycholic acid 3,12-diacetate (5b): Yield 90%; white solid, mp 125–126 °C (lit.
2
5
1
colorless oil); [α]
D
+ 65.5 ° (c 1.0, CHCl
3
) (lit 68.6 °). H NMR (CDCl
3
, 500 MHz) δ 5.07
(
2
1H, dd, J = 3.0, 2.5 Hz, H-12), 4.69 (1H, tt, J = 11.0, 4.2 Hz, H-3) , 2.37 (1H, m, H-23b),
.24 (1H, m, H-23a), 2.09 (3H, s, COCH ), 2.02 (3H, s, COCH ), 0.90 (3H, d, J = 6.4 Hz,
H-21), 0.89 (3H, s, H-19), 0.71 (3H, s, H-18); C NMR (CDCl , 500 MHz) δ 179.7 (C-24),
70.6 (COCH ), 170.5 (COCH ), 75.9 (C-12), 74.2 (C-3), 30.6 (C-23), 23.0 (C-19), 21.4
CO-), 21.3 (CH CO-), 17.5 (C-21), 12.4 (C-18). HRMS: [M + Na] Calcd.
499.3036. Found: C27 499.3041.
3
3
1
3
3
1
(
C
3
3
+
CH
3
3
27
H
44NaO
6
H
44NaO
6
2
5
Deoxycholic acid 12-acetate (6b): Yield 60%; colorless oil; [α]
1
D
+ 40.1 ° (c 1.0, CHCl ).
3
H NMR (CDCl3, 500 MHz) δ 5.07 (1H, t, J = 2.6 Hz, H-12), 3.62 (1H, tt, J = 11.0,4.1 Hz,
), 0.89 (3H, s, H-19),
.82 (3H, d,J = 6.4 Hz, H-21), 0.72 (3H, s, H-18); 13C NMR (CDCl3, 500 MHz) δ178.9
C-24), 170.6 (COCH ), 75.9 (C-12), 71.8 (C-3),30.8 (C-23) ,23.1 (C-19), 17.5 (C-21), 12.4
C-18). HRMS: [M + Na] Calcd. C26
H-3), 2.38 (1H, m, H-23b), 2.23 (1H, m, H-23a), 2.08 (3H,s, COCH
3
0
(
(
3
+
H42NaO
5
457.2930. Found: C28
H
46NaO 457.2934.
5
Ethyl Chenodeoxycholate acid 7-acetate (7a): Yield 93%; white solid, mp 88–89 °C;
2
5
1
[
α]
H-7), 4.11 (2H, q, J = 6.5 Hz, OCH
H-23b), 2.21 (1H, m, H-23a), 2.04 (3H, s, COCH
D
+ 6.5 ° (c 1.0, CHCl
3
). H NMR (CDCl
CH ), 3.49 (1H, tt, J = 11.2, 4.3 Hz, H-3), 2.31 (1H, m,
), 1.24 (3H, t, J = 6.5 Hz, -OCH CH ),
.92 (3H, d, J = 6.5 Hz, H-21), 0.91 (3H, s, H-19), 0.64 (3H, s, H-18); C NMR (CDCl
00 MHz) δ174.3 (C-24), 170.6 (COCH ), 71.8 (C-7), 71.3 (C-3), 60.2 (OCH CH ), 30.8
CO-), 18.3 (C-21), 14.2 (OCH CH ), 11.7 (C-18). HRMS:
485.3243. Found: C28 485.3238.
3
, 500 MHz) δ 4.86 (1H, dd, J = 3.0, 2.5 Hz,
2
3
3
2
3
1
3
0
5
(
[
3
,
3
2
3
C-23), 22.7 (C-19), 21.6 (CH
3
2
3
+
M + Na] Calcd. C28
H46NaO
5
H46NaO
5
4
.6. Drug Screening
Trypanosoma cruzi epimastigotes from clone CL Brener were grown at 28 °C in liver
infusion tryptose (LIT) medium supplemented with 10% heat-inactivated fetal bovine
serum, 20 mg/ml of hemin, 100 µg/ml of streptomycin and 100 U/ml of penicillin.
6
Epimastigotes were seeded at a final concentration of 5 × 10 cells/mL and were incubated
with different concentrations of the compounds ranging from 0.14 µM to 300 µM during 12
days at 28°C. All drugs tested and benznidazole were dissolved in dimethyl sulfoxide
(
DMSO) and the final DMSO concentration in LIT never exceeded 0.5% (v/v). Control
conditions of parasites without drug (100% growth) and medium without parasites were
included. Parasite viability was determined at 600nm in ELISA reader everyday.
4
.7. Modecular modeling

CAL
http://www.rcsb.org/pdb/), PDB code for CAL B is 1TCA. All substrates were minimized
using semiempirical AM1 method with the algorithm Polak-Ribiere in Hyperchem. The
B
structure
was
downloaded
from
RCSB
Protein
DataBank
(
4
1
docking calculations were carried out with Autodock 4.2 program. The Autodock 4.2
method was applied considering a pair of rotatable bonds for steroids and all the protein
were defined it was considered stiff. For the location and extent of the 3D area, the search
space is defined by specifying a center, the number of points in each dimension, and points
between spaces to focus the search space in the active site of the enzyme, it was taken as
grid box center the coordinates x, y, z of a water molecule close to Ser105.
Supporting Information (see footnote on the first page of this article): Spectral data for
compounds 2-7, associated with this article are supplied.
Acknowledgments
We thank UBA X010, UBACYT 20020100100304, CONICET PIP 112-200801-00801/09,
ANPCyT PICT 2011-00595 and Bunge y Born Foundation for partial financial support.
A.B., S.L. and G.L.L. are Research Members of CONICET (Argentina). The authors
gratefully acknowledge Lic. M. M. Rivero for her assistance in GC analysis and Maprimed
S.A. for the generous gift of benznidazole.
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4

Graphical Abstract
Enzymatic synthesis of bile acid derivatives and
biological evaluation against Trypanosoma cruzi
Leave this area blank for abstract info.
a
a
b
b
a
Guadalupe García Liñares , M. Antonela Zígolo , Leandro Simonetti , Silvia A. Longhi and Alicia Baldessari ∗
Laboratorio de Biocatálisis. Departamento de Química Orgánica y UMYMFOR, Facultad de Ciencias Exactas y Naturales,Universidad de Buenos Aires,
a
b
Ciudad Universitaria,Pabellón 2, piso 3, C1428EGA Buenos Aires, Argentina Instituto de Investigaciones en Ingeniería Genética y Biología Molecular
“Dr. Héctor N. Torres”, CONICET,Vuelta de Obligado 2490, C1428ADN Buenos Aires, Argentina
O
3
R
BILE ACIDS
4
OR
1
R
2
R