An efficient enzymatic preparation of 20-pregnane succinates: chemoenzymatic synthesis of 20β-hemisuccinyloxy-5αH-pregnan-3-one

Article abstract of DOI:10.1016/j.tet.2007.12.006

Lipase-catalyzed transesterification of the 20 hydroxyl group in a series of pregnanes afforded novel 20-ethyl succinates that are not possible to prepare following the traditional synthetic methods. The reaction is stereoselective. The enzyme reacts selectively with the 20β epimers therefore only the 20β-succinyloxy derivatives are obtained. These compounds are obtained in variable yield, depending on the substitution in the ring A. The enzymatic approach allowed, for the first time, the synthesis of 20β-hemisuccinyloxy-5αH-pregnan-3-one, novel compound useful as a precursor of steroid-protein conjugates.

Full text of DOI:10.1016/j.tet.2007.12.006

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 1721e1730
www.elsevier.com/locate/tet
An efficient enzymatic preparation of 20-pregnane succinates:
chemoenzymatic synthesis of 20b-hemisuccinyloxy-
5aH-pregnan-3-one
Leandro N. Monsalve a, Mayra Y. Machado Rada b, Alberto A. Ghini b, Alicia Baldessari a,
*
a Laboratorio de Biocata´lisis, Departamento de Quimica Orga´nica y UMYMFOR, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, C1428EGA Buenos Aires, Argentina
b Departamento de Quimica Orga´nica y UMYMFOR, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, C1428EGA Buenos Aires, Argentina
Received 9 September 2007; received in revised form 5 October 2007; accepted 4 December 2007
Available online 8 December 2007
Abstract
Lipase-catalyzed transesterification of the 20 hydroxyl group in a series of pregnanes afforded novel 20-ethyl succinates that are not possible
to prepare following the traditional synthetic methods. The reaction is stereoselective. The enzyme reacts selectively with the 20b epimers there-
fore only the 20b-succinyloxy derivatives are obtained. These compounds are obtained in variable yield, depending on the substitution in the ring
A. The enzymatic approach allowed, for the first time, the synthesis of 20b-hemisuccinyloxy-5aH-pregnan-3-one, novel compound useful as
a precursor of steroideprotein conjugates.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Candida antarctica lipase B; 20-Pregnane succinates; Enzymatic stereoselective transesterification
1. Introduction
Erlanger et al. have demonstrated, by using deoxycorticoster-
one 21-hemisuccinate and bovine serum albumin (BSA), that
20 NH2 groups over 60 were substituted giving an efficacy
of 33.33%.4 In addition, when progesterone-11a-hemisucci-
nate-BSA was used as conjugate, 36 steroid molecules per pro-
tein molecule were found.5 The conjugates can be prepared by
linking the steroid hemisuccinate via an amide bond to the
3-amino groups of lysine in bovine serum albumin (BSA) by
means of a mixed anhydride intermediate.6
Regarding the steroid, in the case of dexamethasone the
preparation of 21-hemisuccinate is easy and the product is
obtained in high yield. It is generally performed by reacting
dexamethasone with excess of succinic anhydride and 4-
dimethylaminopyridine for 24 h at room temperature.7 Hemi-
succinate derivatives of several steroids have been reported
and some of them are commercial products. For example,
there are several examples of application of hemiesters of
succinic acid in different positions of the steroid skeleton. In
the position 3: cholesterol hemisuccinate8a and pregnenolone
hemisuccinate;8b 6: progesterone-6b-hydroxy-hemisuccinate;8c
Steroideprotein conjugates show a wide range of applica-
tions. They are used in affinity chromatography to isolate
proteins with specific functions such as steroid receptors,1
and in new strategies of controlled drug release, for example,
the selective intracellular delivery of the anti-inflammatory
drug, dexamethasone, into endothelial cells.2
Steroideprotein conjugates are also used to obtain antihor-
monal agents. In the field of Endocrinology, the antihormonal
agents capable of counteracting the physiological effects of
endogenous hormones could serve as important biochemical
and clinical tools.3
Several papers have previously described the efficacy of
succinyl-substituted steroids as conjugates. For instance,
* Corresponding author. Fax: þ54 11 4576 3385.
E-mail addresses:
monsalve@qo.fcen.uba.ar
(L.N. Monsalve),
yosymachado@qo.fcen.uba.ar (M.Y. Machado Rada), ghini@qo.fcen.uba.ar
(A.A. Ghini), alib@qo.fcen.uba.ar (A. Baldessari).
0040-4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.12.006

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L.N. Monsalve et al. / Tetrahedron 64 (2008) 1721e1730
11a: progesterone-11a-hydroxy-hemisuccinate;8d 17: medroxy-
progesterone-17-hemisuccinate8e and 17b-estradiol-17-hemi-
succinate,8f and mostly in the position 21: the above mentioned
dexamethasone-21-hemisuccinate,6 6a-methylprednisolone-21-
hemisuccinate,8g hydrocortisone-21-hemisuccinate,8h 6b-hy-
droxycortisol-21-hemisuccinate,8i etc.
R2
R1
R3
R4
R5
However, the approach used to prepare the above men-
tioned hemisuccinates involved the esterification of the
hydroxyl group in position 20 of pregnanes but did not afford
satisfactory results. Since pregnane-20-hemisuccinates are
required as starting material in the preparation of steroidepro-
tein conjugates that could be useful, inter alia in the study of
the relationship between steroidogenesis and spermatogenesis
in male amphibians in vivo,9 it is desirable to have an efficient
method for their preparation. With these guidelines in mind,
we decided to apply an enzymatic approach.
The use of enzymes in the synthesis of pharmaceuticals and
natural products’ derivatives is increasing in the last few
years.10,11 Biotransformation is an area of interest for the prep-
aration of new compounds with added value to the physiologi-
cal and therapeutic properties, which are difficult to obtain by
conventional chemical methods.12
In the last years, lipases have become attractive as biocata-
lysts for chemo-, regio-, and stereoselective reactions under
mild conditions.13 They can be used in a wide variety of
organic solvents and do not require a coenzyme for activity.14
Specifically in the steroid field, enzyme catalysis can play
an important role in the mild and selective interconversion of
functional groups via regioselective transformations.15e18
Studies carried out in our laboratory on the esterification and
transesterification of polyfunctionalyzed steroids have shown
that lipases can act on substituents either on A-ring or on the
D-ring.19,20 In addition, in previous papers we observed that,
in androstanes and pregnanes, Candida rugosa lipase showed
a preference for C-3 hydroxyl or acyloxy groups, whereas
Candida antarctica catalyzed the reactions in D-ring.21 Taking
into account these properties we have prepared fatty acid de-
rivatives of dehydroepiandrosterone22 and 3,17-b-estradiol.23
As a part of our ongoing project on enzymatic transforma-
tion of steroids, we report in the present paper that the results
obtained by the application of a lipase-catalyzed reaction in
the preparation of a series of novel 20-succinates of the preg-
nanes 1de6d (Fig. 1).
1a: R1 = AcO ; R2 = -α-OH ; R3, R4 = Δ5
-
-
1b: R1 = AcO ; R2 = -β-OH ; R3, R4 = Δ5
1c: R1 = AcO ; R2 = -α-OCO(CH2)2COOEt ; R3, R4 = Δ5
-
1d: R1 = AcO ; R2 = -β-OCO(CH2)2COOEt ; R3, R4 = Δ5
-
1e: R1 = HO ; R2 = -β-OH ; R3, R4 = Δ5
-
1f: R1 = HO ; R2 = -β-OCO(CH2)2COOEt ; R3, R4 = Δ5
-
2a: R1 = t-BuMe3Si ; R2 = -α-OH ; R3, R4 = Δ5
-
2b: R1 = t-BuMe3Si ; R2 = -β-OH ; R3, R4 = Δ5
-
2c: R1 = t-BuMe3Si ; R2 = -α-OCO(CH2)2COOEt ; R3, R4 = Δ5
-
2d: R1 = t-BuMe3Si ; R2 = -β-OCO(CH2)2COOEt ; R3, R4 = Δ5
-
3a: R1 = AcO ; R2 = -α-OH ; R3 = R4 = H
-
2
3b: R1 = AcO ; R = -β-OH ; R3 = R4 = H
-
2
3c: R1 = AcO ; R = -α-OCO(CH2)2COOEt ; R3 = R4 = H
-
3d: R1 = AcO ; R2 = -β-OCO(CH2)2COOEt ; R3 = R4 = H
-
4a: R1 = -O(CH2)2O ; R2 = -α-OH ; R3, R5 = Δ5
-
4b: R1 = -O(CH2)2O ; R2 = -β-OH ; R3, R5 = Δ5
-
4c: R1 = -O(CH2)2O ; R2 = -α-OCO(CH2)2COOEt ; R3, R5 = Δ5
-
4d: R1 = -O(CH2)2O ; R2 = -β-OCO(CH2)2COOEt ; R3, R5 = Δ5
-
5a: R1 = O= ; R2 = -α-OH ; R3, R5 = Δ4
5b: R1 = O= ; R2 = -β-OH ; R3, R5 = Δ4
5c: R1 = O= ; R2 = -α-OCO(CH2)2COOEt ; R3, R5 = Δ4
5d: R1 = O= ; R2 = -β-OCO(CH2)2COOEt ; R3, R5 = Δ4
6b: R1 = O= ; R2 = -β-OH ; R3 = R4 = H
6d: R1 = O= ; R2 = -β-OCO(CH2)2COOEt ; R3 = R4 = H
7: R1 = O= ; R2 = -β-OCO(CH2)2COOH ; R3 = R4 = H
Figure 1. 20-Hydroxy-pregnane and its corresponding succinates.
as substrate and succinic anhydride as acylating agent, taking
into account its good performance in the subtilisin-catalyzed
esterification of pregnanes in the position 3 of ring A.24 We
studied the esterification catalyzed by several commercially
available lipases: C. antarctica lipase B (CAL B), Pseudomo-
nas cepacia lipase (PSL), C. rugosa lipase (CRL), porcine
pancreatic lipase (PPL), and Rhizomucor miehei lipase (LIP),
in a variety of solvents (t-amyl alcohol, acetonitrile, acetone,
diisopropyl ether, chloroform, hexane, toluene, and tetrahydro-
furan). None of the enzymes tested catalyzed the reaction with
1b, and similar conditions applied to 1a, 2a, 2b, 6a, and 6b
gave the same unsatisfactory results. The only result obtained
with succinic anhydride and 1b was observed when we used
CRL as biocatalyst and diisopropyl ether as solvent, but the
product was the diol 1e in 5% yield (Table 1, entry 1).
Using ethyl hemisuccinate instead of succinic anhydride
and CAL B and CRL as biocatalysts (entries 2 and 3), it
was possible to obtain the desired product 1d but in very
low amounts and accompanied by a mixture of steroid prod-
ucts such as 1e and 1f, together with diethyl succinate and suc-
cinic acid.
Encouraged by our success in lipase-catalyzed esterifica-
tion in 20-hydroxypregnanes, we applied this methodology
to the synthesis of 20b-hemisuccinyloxy-5aH-pregnan-3-one
(7) (Fig. 1). In this paper we also report for the first time a
chemoenzymatic way to prepare 7 that will allow determina-
tion of the level of circulating steroids during the reproductive
season in male toads.9
2. Results and discussion
In summary, under these reaction conditions, neither suc-
cinic anhydride nor ethyl hemisuccinate proved to be good
acylating agents.
2.1. Enzymatic reactions
In view of the previous satisfactory results that we had
obtained in enzymatic transesterification reactions,25,26 we
screened diethyl succinate as acylating agent and 1b as
Our initial experiments to obtain the 20-succinates were
performed using 3b-acetyloxy-20b-hydroxypregn-5-ene (1b)

L.N. Monsalve et al. / Tetrahedron 64 (2008) 1721e1730
1723
Table 1
Lipase-catalyzed transesterification of pregnanesa
Entry
Substrate
Enzyme
Solvent
Acylating agent
Product (% yield)
1
1b
1b
1b
1b
1b
1bb
1b
1a
2a
2b
3a
3b
4a
4b
5a
5b
6b
CRL
CAL B
CRL
DIPE
DIPE
Succinic anhydride
Ethyl hemisuccinate
Ethyl hemisuccinate
Diethyl succinate
Diethyl succinate
Succinic anhydride
Diethyl succinate
Diethyl succinate
Diethyl succinate
Diethyl succinate
Diethyl succinate
Diethyl succinate
Diethyl succinate
Diethyl succinate
Diethyl succinate
Diethyl succinate
Diethyl succinate
1e (5)
1d (6)
2
3
DIPE
Hexane
1d (9)þ1e (10)þ1f (21)
4
CAL B
CAL B
CAL B
CRL
1d (20)þ1e (5)þ1f (12)
5
Isooctane
Isooctane
Isooctane
Isooctane
Isooctane
Isooctane
Isooctane
Isooctane
Isooctane
Isooctane
Isooctane
Isooctane
Isooctane
1d (43)
1d (39)
d
6
7
8
CAL B
CAL B
CAL B
CAL B
CAL B
CAL B
CAL B
CAL B
CAL B
CAL B
d
d
9
10
11
12
13
14
15
16
17
a
2d (38)
d
3d (42)
d
4d (30)
d
5d (72)
6d (77)
Reaction conditions: E/S 5:1, A/S 3:1; temperature: DIPE and hexane: 55 ꢀC, isooctane: 100 ꢀC; reaction time: isooctane: 2 h, DIPE and hexane: 120 h.
One-pot procedure.
b
substrate. The lipase from C. antarctica B (CAL B) with hex-
was observed. Considering non-polar solvents, if we compare
hexane and isooctane the difference in polarity among them is
low (log P hexane: 3.5 and log P isooctane: 4.5),30 showing
that the difference in solvent polarity alone does not explain
the increase in reaction regioselectivity. Since in isooctane
the reaction temperature is 45 ꢀC higher than in hexane, we
suggest that the increase in regioselectivity of enzymatic be-
havior could be associated with a kinetically controlled
process.
In view of the promising results obtained in the enzymatic
preparation of 20-ethyl succinate of 1b, we decided to apply
the same reaction conditions in the preparation of a series of
pregnane-20-succinates. The results are summarized in Table
1 (entries 9e17). In every case the enzymatic transesterifica-
tion was clean and the 20-ethyl succinates were the only deriva-
tives obtained.
ane as solvent gave the best results (1d in 20% yield) (entry 4),
although not selective because 3b,20b-dihydroxypregn-5-ene
(1e) and 20b-ethylsuccinoyloxy-3b-hydroxypregn-5-ene (1f)
were also obtained in 5% and 12% yield, respectively. LIP
showed a lower performance and the other lipases (PPL,
PSL, and CRL) were completely inert.
In order to improve the activity of CAL B, we decided to
perform the reactions at higher temperature and carried out
the transesterification using three non-polar solvents: hexane
(bp: 69 ꢀC), toluene (bp: 110 ꢀC), and isooctane (2,2,4-trime-
thylpentane) (bp: 99 ꢀC) at their boiling temperatures. The
best results were obtained with isooctane giving 1d as the
only product in 43% yield.
The succinoylation of 1b on the hydroxyl of carbon 20 was
1
confirmed by observing the shift in the H NMR signal of
H-20 resonance from 3.73 ppm in substrate 1b to 4.86 ppm in
product 1d. There is also a significant difference in methyl-
18 chemical shift, which moves from 0.77 ppm to 0.64 ppm.
In order to optimize the reaction conditions in isooctane,
we performed several experiments varying other reaction pa-
rameters such as enzymeesubstrate ratio (E/S) and acylating
agentesubstrate ratio (A/S). We have chosen as standard con-
ditions diethyl succinate as acylating agent, isooctane as sol-
vent, reflux temperature, an E/S ratio of 5 and A/S ratio of 3.
It is remarkable the variation in regioselective behavior dis-
played by the lipase (1dþ1eþ1f, Table 1, entry 4) in the trans-
esterification reaction with diethyl succinate using hexane as
solvent at 55 ꢀC, in contrast with isooctane at 100 ꢀC where
1d was the only product obtained. Many reports demonstrated
that organic media not only influence the enzymatic activity
but also the selectivity,27 and many examples have been
observed about the correlation of enzyme regioselectivity
with solvent parameters,28 especially with hydrophobicity de-
scribed by log P.29 In this work, when polar solvents such as
tetrahydrofurane or acetonitrile were used, no transacylation
The yield was variable and seemed to be dependent more
on the substituent at carbon 3 of ring A than on the presence
of the double bond in ring A or B. The best yields were ob-
tained with substrates 5b (72%) and 6b (77%) containing a car-
bonyl group in the position 3 of ring A. The carbonyl is
a sterically smaller group in comparison with tert-butyldime-
thylsilyl group in 2b and ethylendioxy group in 4b, which
afforded the lowest yields, 38% and 30%, respectively. The
low influence of a double bond was observed in the similar
yields obtained using the 3b-acetates 1b (43%) with a double
bond in position 5 and 3b (42%) saturated in this position.
Similar results can be observed for the substrates 5b and 6b,
which differ in the double bond in 4 and afford 5d and 6d
in almost the same yield.
The stereoselectivity of the enzymatic reaction, which only
produces the 20b-succinyloxy derivative, deserves a special
comment.
As can be observed in Table 1 (entries 9e18) from both ste-
reoisomers in the 20 hydroxyl substrates, only the 20b isomer
was a good substrate in the enzymatic transesterification with

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L.N. Monsalve et al. / Tetrahedron 64 (2008) 1721e1730
CAL B and isooctane at reflux. Under these conditions, only
the desired 20b-ethyl succinate was obtained, whereas the
20a one was inert to the enzymatic reaction in every case.
These results reveal a new type of stereoselective behavior
of CAL B, which has been rationalized by molecular model-
ing.31 We have also examined these enzymatic reactions using
molecular modeling, in order to understand the structural rela-
tion between the enzymatic pocket and the characteristic ste-
roidal framework, which accounts for the remarkable
selectivity of the biological behavior of these compounds. In
fact, only the 20b isomers have biological activity and would
be useful in the preparation of steroideprotein conjugates.
By docking the substrates, we observed that the most favor-
able orientation for the C-20 hydroxyl respect to the acyleSer
105 complex resulted locating the A-ring of the steroidal skel-
eton deep inside the pocket, near the turn formed by residues
38e44 (Pro-Gly-Thr-Gly-Thr-Thr-Gly). Trp 104 locates close
to steroidal skeleton, forcing methyl groups 18 and 19 to point
to the opposite face. An appropriate trajectory for hydroxyl
group attached to carbon 20 is needed in order to react with
the acyleenzyme complex, which is pointing to the plane
formed by carbonyl group and oxygen of the ester moiety,
opposite to His 224 in the triad. The difference in reactivity
between 20a- and 20b-hydroxy epimers could be explained
by the position of methyl 21 necessary to release the acyleen-
zyme complex. The former shows steric interaction with Ile
˚
189 (distance to residue is about 3 A) whereas the latter seems
to be less impeded to reach a reactive conformation (the sub-
stituent on carbon 20 closest to the residue is a hydrogen). Fig-
ure 2a and b shows the substrates 5a and 5b, together with
acyleSer 105 and other highlighted residues that fix their
position into the catalytic pocket.
Figure 2. (a) Docking of compound 5a. Trp 104 (white) and Ile 189 (red) make
all methyl groups point below the steroidal rings. In this case, hydroxyl on car-
bon 20 is located opposite to Ser 105eacyl complex (brown) and the reaction
cannot be achieved. Ser 47 (gray) shows a favorable interaction with carbonyl
oxygen attached to carbon 3. Residues 38e44 (green backbone) are located
near to the A-ring and interact sterically with bulky substituents at 3b position.
(b) Docking of compound 5b. For this configuration, hydroxyl on carbon 20
points toward the catalytic triad, which explains selectivity for 20-(R)-hydroxy-
pregnanes transesterification.
The molecular modeling also explained the effect of bulky
substituents attached to A-ring, such as 3-acetoxy, 3-tert-butyl-
dimethylsilyl, and 3-ethylenedioxy, which afforded the corres-
ponding products in lower yield. These groups increase the
steric hindrance between residues 38e44 and substrate. How-
ever, it could be assumed that the steric hindrance is not the
only cause of a decrease in enzymatic activity and also elec-
tronic effects from carbonyl group would be related to reaction
yield. For 3-oxo compounds a favorable hydrogen bond inter-
action between the oxygen belonging to the carbonyl group
and the hydroxyl of Ser 47 was observed, which would explain
the increase in yield for these compounds (5d and 6d).
On the other hand, the transesterification reaction of the
3b,20b-diol 1e with CAL B transesterification in isooctane
at reflux did not afford any product. As its solubility in the
reaction system is the same as 6b and the steric hindrance
of hydroxyl group is not higher than a carbonyl, electronic
effects must also be important in the enzymatic reaction.
At this point we could prepare both the acylating agent di-
ethyl succinate and a series of 20b-pregnane ethyl succinates
using a biocatalytic approach. This fact prompted us to apply
a one-pot enzymatic procedure32 in which the acylating agent
and the ethyl succinate of 1b were obtained in the same step.
Therefore, we treated 1b with succinic anhydride, ethanol,
and isooctane as solvent in the presence of CAL B. The
reaction was performed under reflux and with an E/S ratio
of 5, a succinic anhydride/1b ratio of 3, and a succinic anhy-
dride/alcohol ratio of 1. It was observed that the ethyl deriva-
tive 1d was obtained in similar yield (39%) as in the
enzymatic transesterification with diethyl succinate as acyl-
ating agent (43%), so the one-pot procedure could be consid-
ered as an alternative method for the preparation of the
succinyloxy derivatives.
Finally, the lipase is recyclable. As it is insoluble in the
organic reaction media it is easily removed by filtration at
the end of the process. It can be re-used and in this particular
reaction, CAL kept 80% activity after eight reaction cycles.
2.2. Synthesis of enzyme substrates
Pregnane derivatives 1e5 were synthesized from pregneno-
lone acetate using standard procedures previously reported by
us.33,34

L.N. Monsalve et al. / Tetrahedron 64 (2008) 1721e1730
1725
2.3. Chemoenzymatic synthesis of 20b-hemisuccinyloxy-5aH-
pregnan-3-one
in 77% yield. Finally, compound 7 was obtained by regioselec-
tive cleavage of the ethyl group with bis(tributyltin)oxide
following the procedure described by Mascaretti et al.37
The enzymatic procedure was applied to the preparation of
20b-hemisuccinyloxy-5a-pregnan-3-one (7), not previously
reported (Scheme 1). The substrate 20b-hydroxy-5a-preg-
nan-3-one (6b) was also prepared in our laboratory. In the
synthetic route to 6b we used as intermediate 3b,20b-dihy-
droxy-5a-pregnane 10.35 It was obtained from commercially
available pregnenolone acetate (8) in three steps using stan-
dard procedures. Thus, catalytic hydrogenation of the D5
double bond in 8, following by simultaneous reduction of 10
of 20-carbonyl moiety and deprotection of 3-acetate 9 with
LAH in THF rendered in 91% yield (three steps). The synthetic
sequence is outlined in Scheme 1. Formylation of the two hy-
droxyl groups in 10 was accomplished using mixed formic ace-
tic anhydride36 rendering diformiate 11 in almost quantitative
yield. Characteristic signals of the formyl protons and carbons
in the NMR of 11 were especially diagnostic of the success of
this reaction. Regioselective deformylation of 11 (KCO3H/
MeOH) and further oxidation with pyridinium chlorochromate
(PCC) furnished the 3-keto-20-formyloxy pregnane 12 in 85%
yield (from 11). Spectroscopic evidence for this compound was
the absence of a signal for a hydrogen at C-3 in the 1H NMR and
the signal at 204 ppm in the 13C NMR characteristic of a 3-keto
pregnane. Clean hydrolysis of the 20-formate in 12 was easily
accomplished by treatment with KOH/MeOH, giving rise to
3-keto-20-hydroxy derivative 6b in quantitative yield. The
above mentioned CAL B-catalyzed transesterification of 6b
with ethyl succinate afforded the succinoylated derivative 6d
3. Conclusions
Enzymatic catalysis allows the transesterification of the 20
hydroxyl group in a series of pregnanes affording novel 20-
alkyl succinates (1de6d) which are not possible to prepare
following the traditional synthetic methods. Through a simple
hydrolysis of these compounds 1de6d, it is possible to obtain
various hemisuccinate derivatives. These derivatives can be
useful as substrates in the synthesis of steroideprotein conju-
gates. It is important to emphasize the completely stereoselec-
tive behavior of the lipase. Only the 20b epimer acts as
substrate of the enzymatic reaction. As a result the 20b-ethyl
succinates are exclusively obtained. It was also observed that
the yield depended on the substitution in carbon 3 of ring A.
These experimental facts have been verified by docking of
the substrates into the catalytic pocket of CAL B lipase.
This biotechnological procedure shows several advantages
such as mild reaction conditions and low environmental
impact. Moreover, as enzyme is insoluble in the reaction me-
dium, it is easily removed by filtration at the end of the process
and can be re-used. In this particular reaction CAL B keeps
80% of its activity after eight reaction cycles. Finally, this
work also describes the application of the enzymatic approach
to the first synthesis of 5a-pregnane-3-one-20b-hemisuccinate
7, a novel compound with potential application in the synthe-
sis of antihormonal steroideprotein conjugates.
O
O
OR2
b, c
a
AcO
AcO
R1O
H
H
10 : R1 = R2 = H
8
9
11 : R1 = R2 = CHO
OCHO
OH
d, e
f
O
O
H
H
12
6b
g
OCO(CH2)2COOEt
OCO(CH2)2COOH
h
O
O
H
H
7
6d
Scheme 1. Reagents and conditions: (a) H2/Pd; (b) LAH, THF; (c) HOCOCOCH3, Py; (d) KCO3H, MeOH; (e) PCC, BaCO3, Cl2CH2; (f) KOH, MeOH; (g) CAL B
lipase, diethyl succinate, isooctane, reflux; (h) bis(tributyltin)oxide, toluene.

1726
L.N. Monsalve et al. / Tetrahedron 64 (2008) 1721e1730
1
4. Experimental
4.1. General remarks
into account the integration of the CH3-13 signal in their H
NMR spectrum) was separated by chromatography on silica
gel with hexaneeethyl acetate as eluent. 1H NMR
(200.13 MHz): 1a: d 0.68 (3H, s) (Me-18), 1.03 (3H, s)
(Me-19), 1.23 (3H, d, J¼6.0 Hz) (Me-21), 2.03 (3H, s), 3.73
(1H, m) (H-20), 4.62 (1H, br s) (H-3), 5.39 (1H, dd) (H-6);
1b: d 0.78 (3H, s) (Me-18), 1.04 (3H, s) (Me-19), 1.14 (3H, d,
J¼6.1 Hz) (Me-21), 3.72 (1H, m) (H-20), 4.58 (1H, br s)
(H-3), 5.36 (1H, dd) (H-6).
Lipase from C. rugosa (CRL) (905 U/mg solid) and type II
crude from porcine pancreas (PPL) (190 U/mg protein) were
purchased from Sigma Chemical Co.; lipase from P. cepacia:
Lipase PS-C Amano II (804 U/g) was purchased from Amano
Pharmaceutical Co. C. antarctica lipase B (CAL B): Novo-
zymÒ 435 (7400 PLU/g) and R. miehei lipase: Lipozyme
RM 1M (LIP) (7800 U/g) were generous gifts of Novozymes
Latinoamerica Ltda. and Novozymes A/S. All enzymes were
used ‘straight from the bottle’. Chemical reagents and solvents
were commercialized by Aldrich, Sigma, Fluka, or J.T. Baker.
Enzymatic reactions were carried out on Innova 4000 digital
incubator shaker, New Brunswick Scientific Co. at 200 rpm.
Melting points were measured in a Fisher Johns apparatus
and are uncorrected. Reactions were followed by TLC on
Merck Silica gel 60F-254 aluminum sheets (0.2 mm thick-
ness). For flash chromatography Merck Silica gel 60 (60e
230 mesh) was used. Optical rotations were measured using
chloroform as solvent with a Perkin Elmer 343 polarimeter.
FTIR spectra were obtained on a Shimadzu FTIR-8300 spec-
4.3.2. 3b-tert-Butyldimethylsilyloxy-20a-hydroxypregn-5-ene
(2a) and 3b-tert-butyldimethylsilyloxy-20b-hydroxypregn-
5-ene (2b)
These compounds were obtained from pregnenolone ace-
tate by reduction with LAH that rendered the 3,20-dihydroxy-
1
pregne-5-ene as a mixture of 20a/20b epimers (1:9 by H
NMR) in almost quantitative yield. The mixture was dissolved
in anhydrous DMF (20 mL) under nitrogen, imidazole
(3 mmol) and tert-butyldimethylsilyl chloride (2 mmol) were
added at 0 ꢀC and the mixture was allowed to reach 25 ꢀC. Af-
ter 2 h the solution was poured into saturated aqueous NaCl
(60 mL), extracted with diethyl ether (3ꢁ10 mL), dried with
sodium sulfate, and evaporated under vacuum. The white solid
obtained was purified by flash chromatography on silica gel
using hexane/ethyl acetate (90:10), to give the title compound
2b (Mp: 153e154 ꢀC) in 78% yield. Continued elution with
the same solvent afforded crystalline compound 2a in 13%
yield (Mp: 164e165 ꢀC). 2a: IR (film, cmꢂ1): 3406, 2962,
1
trophotometer. H NMR and 13C NMR spectra were recorded
in CDCl3 as solvent using a Bruker AC-200 spectrometer
1
operating at 200.13 MHz and 50.32 MHz for H and 13C, res-
pectively, and a Bruker AM-500 NMR instrument operating at
500.14 MHz and 125.76 MHz for H and 13C, respectively.
1
1
Chemical shifts are reported in d units relative to tetramethyl-
silane (TMS) set at 0 d, and coupling constants are given in
hertz.
2847, 1454, 1245, 1120, 961, 687; H NMR (500.14 MHz):
d 0.06 (6H, s) (MeeSi), 0.68 (3H, s) (Me-18), 0.89 (9H, s)
(t-BueSi), 1.00 (3H, s) (Me-19), 1.23 (3H, d, J¼6.2 Hz)
(Me-21), 3.71 (1H, m) (H-20), 3.48 (1H, m) (H-3), 5.32 (1H,
br d) (H-)6; 13C NMR (50.32 MHz): d ꢂ4.6, 12.5, 18.3, 19.4,
20.8, 23.5, 24.2, 25.9, 26.0, 31.6, 31.9, 32.1, 36.7, 37.5, 38.9,
41.6, 42.8, 50.2, 56.6, 56.0, 58.4, 70.4, 72.6, 121.1, 141.6.
Anal. Calcd for C27H48O2Si: C, 74.94%; H, 11.18%. Found:
C, 75.06%; H, 11.04%. 2b: IR (film, cmꢂ1): 3390, 2987,
4.2. Molecular modeling
CAL B structure was downloaded from RCSB Protein Data
Bank (http://www.rcsb.org/pdb/).38 Acyl moiety was added to
Ser 105 and the geometry of the resulting complex was ori-
ented according to data published by Pleiss et al.39 and opti-
mized using AMBER algorithm included on HyperChem
version 7.5. All substrates were minimized using semiempiri-
cal AM1 method integrated on the same software mentioned
above.
Docking experiments were run using GOLD version 2.0
(CCDC, Cambridge, UK). The active site radius was set to
˚
10 A and the GoldScore algorithm was used. Twenty solutions
were obtained for each ligand. Structures in Figure 2 were
generated using VMD 1.8.5.40
1
2852, 1545, 1250, 1180, 964, 688; H NMR (500.14 MHz):
d 0.06 (3H, s) (MeeSi), 0.77 (3H, s) (Me-18), 0.89 (9H, s)
(t-BueSi), 1.01 (3H, s) (Me-19), 1.14 (3H, d, J¼6.1 Hz)
(Me-21), 3.74 (1H, m) (H-20), 3.48 (1H, m) (H-3), 5.31 (1H,
br d) (H-6); 13C NMR (50.32 MHz): d ꢂ4.6, 12.4, 18.3, 19.5,
21.0, 23.7, 24.6, 25.7, 26.0, 26.0, 31.8, 32.0, 32.1, 36.7, 37.4,
40.0, 42.3, 42.9, 50.2, 56.3, 58.5, 70.6, 72.7, 121.0, 141.7.
Anal. Calcd for C27H48O2Si: C, 74.94%; H, 11.18%. Found:
C, 75.10%; H, 10.97%.
4.3.3. 3b-Acetyloxy-20a- and 3b-acetyloxy-20b-hydroxy-
5aH-pregnane (3a and 3b)
4.3. Synthesis of substrates (1ae6a and 1be6b) and
acylating agents
3b-Acetyloxy-20-hydroxypregnane, a and b (3a and 3b)
(identical with authentic standards)35 were obtained from
3b-acetoxypregnan-20-one, 2 in 97% yield, by reduction
with NaBH4 in MeOH/CH2Cl2; the epimeric mixture (w1:9
4.3.1. 3b-Acetyloxy-20a-hydroxypregn-5-ene (1a) and 3b-
acetyloxy-20b-hydroxypregnen-5-ene (1b)
Compounds 1a and 1b (identical with authentic stan-
dards)35 were obtained from pregnenolone acetate (3b-acetyl-
oxypregn-5-en-20-one) in 99% yield, by reduction with
NaBH4 in MeOH/CH2Cl2; the epimeric mixture (w1:9 taking
1
by H NMR) was separated by chromatography on silica gel
with hexaneeethyl acetate as eluent. 1H NMR (500.14 MHz):
3a: d 0.67 (3H, s) (Me-18), 0.83 (3H, s) (Me-19), 1.13 (3H, d,
J¼6.0 Hz) (Me-21), 3.71 (1H, m) (H-20), 4.73 (1H, m) (H-3);

L.N. Monsalve et al. / Tetrahedron 64 (2008) 1721e1730
1727
3b: d 0.73 (3H, s) (Me-18), 0.82 (3H, s) (Me-19), 1.14 (3H, d,
J¼5.9 Hz) (Me-21), 3.72 (1H, m) (H-20), 4.72 (1H, m) (H-3).
(Me-19), 1.13 (3H, d, J¼6.0 Hz) (Me-21), 3.59 (1H, m) (H-3),
3.72 (1H, m) (H-20).
4.3.4. 3,3-Ethylenedioxy-20a- and 3,3-ethylenedioxy-20b-
hydroxy-5-pregnene (4a and 4b)
4.4.2. 3b,20b-Diformyloxy-5aH-pregnane (11)
To a solution of the 3b,20b-pregnanediol (10) obtained
above (390 mg, 1.2 mmol) in dry pyridine (10.0 mL) recently
prepared formic acetic anhydride41 (7 mL) was added at 25 ꢀC
under nitrogen atmosphere. After 2 h the solution was poured
into cold 2 N HCl (50 mL), extracted with dichloromethane
(3ꢁ15 mL), dried with sodium sulfate and the solvent evapo-
rated, affording diformyloxypregnane 11 (438 mg, 97% yield).
Mp: 143e145 ꢀC. IR (film, cmꢂ1): 2928, 1707, 1449, 1177,
Compounds 4a and 4b were obtained from 3-oxo-22,23-
bisnor-4-cholenic acid in three steps.41 Decarboxylation with
iodobenzene diacetate and iodine in fresh CCl4 under irradia-
tion with a 300 W tungsten lamp rendered a mixture of 20a
1
(S)/20b (R) epimers (3:2 by H NMR), which was separated
by chromatography in silica gel (hexane/ethyl acetate) giving
rise to the mixture of two 20-iodo-4-pregnene-3-one 20R
(38%) and 20S (56%). Protection of the 3-carbonyl group as
an ethylene ketal and further replacement of iodine for hy-
droxide, via oxygenation of an intermediate 20-carbon radical,
yielded the titled compounds, identical with an authentic stan-
dard41 (4a: 40% yield, 4b: 22% yield). 1H NMR (200.13 MHz):
4a: d 0.68 (3H, s) (Me-18), 1.03 (3H, s) (Me-19), 1.23 (3H, d,
J¼6.2 Hz) (Me-21), 2.55 (1H, m) (Hb-12), 3.71 (1H, m)
(H-20), 3.94 (4H, m) (OCH2CH2O), 5.34 (1H, br d) (H-6); 4b:
d 0.78 (3H, s) (Me-18), 1.04 (3H, s) (Me-19), 1.15 (3H, d,
J¼6.1 Hz) (Me-21), 2.55 (1H, m) (Hb-12), 3.74 (1H, m)
(H-20), 3.95 (4H, m) (OCH2CH2O), 5.35 (1H, br d) (H-6).
1
1041, 677; H NMR (200.13 MHz): d 0.63 (3H, s) (Me-18),
0.82 (3H, s) (Me-19), 1.20 (3H, d, J¼6.6 Hz) (Me-21), 4.81
(1H, m) (H-3), 4.97 (1H, m) (H-20); 13C NMR (50.32 MHz):
d 12.1, 12.3, 20.9, 23.4, 24.3, 25.6, 28.7, 29.7, 31.5, 33.2,
35.2, 36.2, 39.7, 39.9, 41.5, 44.7, 53.8, 55.6, 58.5, 70.6, 72.4.
Anal. Calcd for C23H36O4: C, 73.37%; H, 9.64%. Found: C,
73.13%; H, 9.71%.
4.4.3. 20b-Formyloxyl-3b-hydroxy-5aH-pregnane (12)
Compound 11 (438 mg) was dissolved in THF (10 mL) and
methanol (50 mL) and potassium bicarbonate (3ꢁ35 mg) was
added over 3 h. The reaction mixture was stirred for 4 h at
25 ꢀC under nitrogen atmosphere, and the solution was neu-
tralized with hydrochloric acid, concentrated and then diluted
with water. Extractive workup with diethyl ether gave the 3b-
hydroxy-20b-formyloxy steroid 12 (387 mg), which was puri-
fied by flash chromatography. Mp: 165e166 ꢀC. IR (film,
cmꢂ1): 3414, 2945, 1732, 1375, 1037, 963; 1H NMR
(200.13 MHz): d 0.63 (3H, s) (Me-18), 0.79 (3H, s) (Me-
19), 1.18 (3H, d, J¼6.2 Hz) (Me-21), 3.59 (1H, m) (H-3),
4.96 (1H, m) (H-20); 13C NMR (50.32 MHz): d 12.4, 12.5,
21.3, 22.8, 24.4, 25.5, 28.6, 31.5, 32.1, 35.5, 36.2, 37.0,
38.2, 39.1, 44.9, 54.4, 55.9, 56.7, 63.9, 71.3, 72.9, 161.0.
Anal. Calcd for C22H36O3: C, 75.82%; H, 10.41%. Found:
C, 75.63%; H, 10.51%.
4.3.5. 20a- and 20b-Hydroxypregn-4-en-3-one (5a and 5b)
Prepared from 4a and 4b by removal of the 3-ethylene ketal
(acetone, p-toluensulfonic acid) giving the corresponding
20-hydroxy-4-pregnen-3-one identical to an authentic standard
(5a: 83% yield, 5b: 82% yield).34 1H NMR (200.13 MHz): 5a:
d 0.70 (3H, s) (Me-18), 1.18 (3H, s) (Me-19), 1.23 (3H, d,
J¼6.3 Hz) (Me-21), 3.72 (1H, m) (H-20), 5.72 (1H, br d)
(H-4); 5b: d 0.78 (3H, s) (Me-18), 1.14 (3H, d, J¼6.3 Hz)
(Me-21), 1.23 (3H, s) (Me-19), 3.72 (1H, m) (H-20), 5.75 (1H,
br d) (H-4).
4.3.6. Preparation of ethyl hemisuccinate
Ethanol (5.8 mL) was added to the solution of succinic an-
hydride (10 g, 100 mmol) in pyridine (20 mL). The reaction
was allowed to proceed in a shaker at 55 ꢀC overnight and
then poured over a water/ice mixture. Hydrochloric acid
(35%) was added dropwise to acidify and the product was
extracted with ethyl acetate. Organic phase was washed with
water, dried, and evaporated. Ethyl hemisuccinate: yield
4.4.4. 20b-Formyloxy-5aH-pregnan-3-one (13)
A suspension of pyridinium chlorochromate (862 mg,
˚
4.0 mmol), barium carbonate (516 mg, 2.6 mmol), and 3 A
molecular sieves (700 mg) in anhydrous dichloromethane
(15 mL) was stirred for 10 min under a nitrogen atmosphere
at 0 ꢀC. A solution of crude 3b-alcohol 12 (387 mg) in anhy-
drous dichloromethane (6 mL) was added and stirring contin-
ued at the same temperature for 2 h. The reaction mixture was
diluted with ether and percolated through Florisil with di-
chloromethane/ether (1:1) and solvent was evaporated. Purifi-
cation of the resulted residue by flash chromatography gave
20-formyloxypregnan-3-one 13 (309 mg, 77% yield, two
steps). Mp: 153e154 ꢀC. IR (film, cmꢂ1): 2932, 1718, 1690,
1434, 1168, 1142, 903; 1H NMR (200.13 MHz): d 0.63 (3H, s)
(Me-18), 1.01 (3H, s) (Me-19), 1.21 (3H, d, J¼6.0 Hz) (Me-
21), 4.96 (1H, m) (H-20); 13C NMR (50.32 MHz): d 12.5,
12.9, 19.8, 21.4, 23.8, 24.6, 28.8, 31.6, 35.1, 35.7, 38.1, 38.4,
39.9, 42.5, 44.8, 46.4, 53.5, 54.8, 55.3, 71.3, 212.3. Anal. Calcd
1
90%, colorless liquid. H NMR (200.13 MHz): d 1.25 (3H, t),
2.65 (4H, m), 4.50 (2H, q); 13C NMR (50.32 MHz): d 13.9,
28.3, 28.5, 59.4, 171.1, 172.7. Anal. Calcd for C6H10O4: C,
49.31%; H, 6.90%. Found: C, 48.89%; H, 6.98%.
4.4. Synthesis of 20b-hydroxy-5aH-pregnan-3-one (6b)
4.4.1. 3b,20b-Pregnanediol (10)
3b,20b-Pregnanediol (10) (Mp: 207e209 ꢀC, identical with
literature)34 was obtained from pregnenolone acetate 8, by cata-
lytic hydrogenation of the double bond following by simulta-
neous reduction of the 20-carbonyl and 3b-acetate moieties
with lithium aluminum hydride in THF (85% overall yield).
1H NMR (200.13 MHz): d 0.74 (3H, s) (Me-18), 0.81 (3H, s)

1728
L.N. Monsalve et al. / Tetrahedron 64 (2008) 1721e1730
for C22H34O3: C, 76.26%; H, 9.89%. Found: C, 79.18%; H,
9.85%.
or at 100 ꢀC with magnetic stirring, and the progress of the re-
action was monitored by TLC (hexane/EtOAc 7:3). Once the
reaction was finished, the enzyme was filtered off and washed
with dichloromethane (3ꢁ5 mL). The organic phases were
combined and dried over Na2SO4. The solvent was removed
under reduced pressure and the crude residue purified by flash
chromatography on silica gel using hexane/EtOAc 95:5.
4.4.5. 20b-Hydroxy-5aH-pregnan-3-one (6b)
20b-Formyloxy-5aH-pregnan-3-one 13 (309 mg) was dis-
solved in THF (5 mL) and methanol (20 mL) and 5% aqueous
potassium hydroxide (2 mL) was added. The resulting solution
was stirred for 5 min at room temperature, neutralized with
2 N hydrochloric acid, and concentrated in vacuo to 1/10 of
the original volume. Dilution with water and conventional
workup with dichloromethane afforded 20b-hydroxy-preg-
nan-3-one 6b (0.253 g, 91% yield). Mp: 185e187 ꢀC. IR
4.6.3. 3b-Acetyloxy-20b-ethylsuccinyloxypregn-5-ene (1d)
White solid (60 mg, 43% yield). Mp: 71e72 ꢀC. [a]D25 ꢂ25.0
(c 0.02, CHCl3). IR (film, cmꢂ1): 2945, 1732, 1375, 1244, 1163,
1035, 963, 903; 1H NMR (200.13 MHz): d 0.64 (3H, s) (Me-18),
1.00 (3H, s) (Me-19), 1.13 (3H, d, J¼6.1 Hz) (Me-21), 1.26 (3H,
t, J¼7.1 Hz) (CH3, OEt), 2.03 (3H, s) (CH3, OAc), 2.59 (4H, m)
(CH2, succinic), 4.14 (2H, q, J¼7.1 Hz) (CH2, OEt), 4.59 (1H,
m, J¼4.2 Hz) (H-3), 4.86 (1H, dq, J¼6.1, 10.5 Hz) (H-20),
5.36 (1H, dd) (H-6); 13C NMR (50.32 MHz): d 12.3, 14.2,
19.3, 19.9, 20.9, 21.4, 24.3, 25.4, 27.7, 29.2, 29.6, 31.7, 31.8,
36.6, 36.9, 38.1, 39.1, 42.1, 50.0, 54.9, 56.0, 60.6, 73.3, 74.0,
122.4, 139.7, 170.6, 171.6, 172.3. Anal. Calcd for C29H44O6:
C, 71.28%; H, 9.08%. Found: C, 71.30%; H, 9.05%.
1
(film, cmꢂ1): 3433, 2926, 171, 1452, 1041, 883; H NMR
(500.14 MHz): d 0.77 (3H, s) (Me-18), 1.02 (3H, s) (Me-19),
1.14 (3H, d, J¼5.9 Hz) (Me-21), 3.73 (1H, m) (H-20); 13C
NMR (50.32 MHz): d 11.6, 12.6, 21.3, 23.7, 24.5, 25.7, 28.9,
31.9, 35.3, 35.7, 38.2, 38.6, 40.0, 42.6, 44.7, 46.7, 53.9, 55.8,
58.6, 70.6, 212.2. Anal. Calcd for C21H34O2: C, 79.19%; H,
10.76%. Found: C, 79.11%; H, 11.11%.
4.5. 20b-Hemisuccinyloxy-5aH-pregnan-3-one (7)
20b-Ethylsuccinyloxy-5aH-pregnan-3-one (6d) (112 mg,
0.25 mmol) was added to a solution of bis(tributyltin)oxide
(0.31 mL, 0.6 mmol) in toluene (7 mL). The mixture was
stirred at 80 ꢀC for 15 h and then the solvent was evaporated
in vacuo. The resulting oil was dissolved in EtOAc (10 mL)
and extracted with 5% aqueous NaHCO3 (3ꢁ5 mL). The
aqueous phase was acidified to pH 4e5 with dilute HCl and
extracted with EtOAc (3ꢁ5 mL). The organic phase was
washed with brine (2ꢁ5 mL), dried (Na2SO4), and evaporated
in vacuo to afford 7 (78 mg, 72% yield). Mp: 208 ꢀC (dec). IR
4.6.4. 3b,20b-Dihydroxypregn-5-ene (1e)
White solid (11 mg, 10% yield). Mp: 210e211 ꢀC (lit.34
210e211 ꢀC). [a]D25 ꢂ65 (c 0.02, CHCl3) (lit.34 ꢂ64); 1H
NMR (200.13 MHz): d 0.77 (3H, s) (Me-18), 1.01 (3H, s)
(Me-19), 1.14 (3H, d, J¼6.1 Hz) (Me-21), 3.50 (1H, m,
J¼4.2 Hz) (H-3), 3.72 (1H, dq, J¼6.1, 10.5 Hz) (H-20), 5.34
(1H, dd) (H-6).
4.6.5. 20b-Ethylsuccinyloxy-3b-hydroxy-pregn-5-ene (1f)
White solid (27 mg, 21% yield). Mp: 112e113 ꢀC. [a]D25
ꢂ23.5 (c 0.02, CHCl3). IR (film, cmꢂ1): 3264, 2936, 2360,
1
(film, cmꢂ1): 3445, 2915, 1727, 1689, 1387, 1036, 871; H
1
NMR (500.14 MHz) for 7: d 0.67 (3H, s) (Me-18), 1.03 (3H,
s) (Me-19), 1.18 (3H, d, J¼6.0 Hz) (Me-21), 1.28 (2H, m)
(H-24), 2.62 (2H, m) (H-23), 4.91 (1H, m) (H-20); 13C
NMR (50.32 MHz): d 11.5, 12.6, 19.9, 21.3, 24.3, 25.5,
28.9, 29.4, 29.7, 31.7, 35.3, 35.7, 38.2, 38.6, 39.3, 42.5,
44.7, 46.8, 53.9, 55.1, 55.7, 73.6, 171.4, 173.6, 212.2. Anal.
Calcd for C25H40O5: C, 71.39%; H, 9.59%. Found: C,
71.23%; H, 9.65%.
2342, 1732, 1449, 1377, 1163, 1054, 1022, 962; H NMR
(200.13 MHz): d 0.64 (3H, s) (Me-18), 0.99 (3H, s) (Me-19),
1.14 (3H, d, J¼6.1 Hz) (Me-21), 1.25 (3H, t, J¼7.1 Hz) (CH3,
OEt), 2.59 (4H, m) (CH2, succinic), 3.51 (1H, m, J¼4.2 Hz)
(H-3), 4.14 (2H, q, J¼7.1 Hz) (CH2, OEt), 4.86 (1H, dq,
J¼6.0, 10.5 Hz) (H-20), 5.34 (1H, dd) (H-6); 13C NMR
(50.32 MHz): d 12.3, 14.2, 19.4, 19.9, 20.9, 24.3, 25.4, 29.2,
29.6, 31.6, 31.9, 36.5, 37.2, 39.2, 42.2, 42.3, 50.1, 55.0, 56.1,
60.6, 71.7, 73.3, 122.5, 140.8, 171.6, 172.3. Anal. Calcd for
C27H42O5: C, 72.61%; H, 9.48%. Found: C, 72.45%; H, 9.50%.
4.6. Enzymatic reactions
4.6.1. Enzymatic preparation of diethyl succinate
4.6.6. 3b-tert-Butyldimethylsilyloxy-20b-ethylsuccinyl-
oxypregn-5-ene (2d)
Diethyl succinate was prepared according to a known pro-
cedure and spectral data were in accordance with those re-
ported in literature.32 Colorless liquid, 88% yield. H NMR
(200.13): d 1.24 (6H, t), 2.60 (4H, s), 4.13 (4H, q).
White solid (51 mg, 38% yield). Mp: 75e77 ꢀC. [a]D25 ꢂ11.9
(c 0.02, CHCl3). IR (film, cmꢂ1): 2930, 2901, 2857, 1732, 1462,
1379,1252,1223,1161,1088,961,887;1HNMR(200.13 MHz):
d 0.05 (6H, s) (MeeSi), 0.63 (3H, s) (H-18), 0.88 (9H, s) (t-Bue
Si), 0.98 (3H, s) (H-19), 1.14 (3H, d, J¼6.1 Hz) (Me-21), 1.26
(3H, t, J¼7.1 Hz), 2.58 (4H, m) (CH2, succinic), 3.47 (1H, m,
J¼4.8 Hz) (H-3), 4.14 (2H, q, J¼7.1 Hz) (CH2, OEt), 4.85
(1H, dq, J¼6.1, 10.5 Hz) (H-20), 5.30 (1H, dd) (H-6); 13C
NMR (50.32 MHz): d ꢂ4.6, 12.3, 14.2, 18.2, 19.4, 19.9, 21.0,
24.3, 25.3, 26.0, 29.1, 29.6, 31.8, 31.9, 32.1, 36.6, 37.4, 39.2,
42.2, 42.8, 50.2, 55.0, 56.1, 60.7, 72.6, 73.3, 120.9, 141.6,
1
4.6.2. General procedure for the enzymatic succinoylation
of pregnanes 1e6
The lipase (500 mg) and the acylating agent (1 mmol) (suc-
cinic anhydride, ethyl hemisuccinate, or diethyl succinate)
were added to a solution of the pregnane 1a/be6a/
b (0.3 mmol) in the corresponding solvent (40 mL). The mix-
ture was incubated in an orbital shaker at 55 ꢀC and 200 rpm

L.N. Monsalve et al. / Tetrahedron 64 (2008) 1721e1730
1729
171.6, 172.3. Anal. Calcd for C33H56O5Si: C, 70.67%; H,
10.06%. Found: C, 70.99%; H, 10.13%.
72.94%; H, 9.07%; O, 17.99%. Anal. Calcd for C27H42O5: C,
72.61%; H, 9.48%. Found: C, 72.84%; H, 9.53%.
4.6.11. Enzymatic one-pot preparation of 3b-ethylendioxy-D5-
pregnen-20b-ethyl succinate (5d)
4.6.7. 3b-Acetyloxy-20b-ethylsuccinyloxy-5aH-pregnane (3d)
White solid (59 mg, 42% yield). Mp: 72e73 ꢀC. [a]D25 þ14.1
(c 0.01, CHCl3). IR (film, cmꢂ1): 2939, 1732, 1375, 1244, 1035,
963, 905, 886; 1H NMR (200.13 MHz): d 0.61 (3H, s) (Me-18),
0.81 (3H, s) (Me-19), 1.15 (3H, d, J¼6.1 Hz) (Me-21), 1.26 (3H,
t, J¼7.1 Hz), 2.02 (3H, s) (CH3, OAc), 2.60 (4H, m) (CH2, suc-
cinic), 3.47 (1H, m, J¼4.8 Hz) (H-3), 4.16 (2H, q, J¼7.1 Hz)
(CH2, OEt), 4.86 (1H, dq, J¼6.1, 10.5 Hz) (H-20); 13C NMR
(50.32 MHz): d 12.2, 14.2, 19.9, 21.1, 21.5, 23.0, 24.2, 25.4,
27.5, 28.5, 32.0, 34.0, 35.3, 35.5, 36.7, 38.7, 39.3, 42.4, 54.2,
55.0, 55.8, 60.6, 73.3, 73.7, 170.7, 171.6, 172.3. Anal. Calcd
for C27H42O5: C, 70.99%; H, 9.45%. Found: C, 71.06%; H,
9.40%.
Succinic anhydride (100 mg, 1 mmol) and 500 mg of CAL
B were added to a stirred solution of compound 5b (108 mg,
0.3 mmol) in 100 mL of ethanol and 40 mL of isooctane.
The mixture was heated at 100 ꢀC and the progress of the re-
action was monitored by TLC (hexane/EtOAc 7:3). Once the
reaction was finished, the enzyme was filtered off and washed
with dichloromethane (3ꢁ5 mL). The organic phases were
combined and dried over Na2SO4. The solvent was removed
under reduced pressure and the crude residue purified by flash
chromatography on silica gel (hexane/EtOAc 95:5).
Acknowledgements
4.6.8. 3,3-Ethylendioxy-20b-ethylsuccinyloxypregn-5-ene (4d)
White solid (41 mg, 30% yield). Mp: 120e122 ꢀC. [a]D25
ꢂ8.1 (c 0.01, CHCl3). IR (film, cmꢂ1): 2939, 2888, 2845,
1730, 1431, 1377, 1313, 1244, 1173, 1153, 1101, 1089, 1029,
We thank Dr. Ernesto Mata for the generous gift of bis(tri-
butyltin)oxide. A.B. thanks UBA (X089) and ANPCyT (PICT
2005, 32735) for partial financial support. A.B. and A.A.G. are
Research Members of CONICET.
1
961, 864; H NMR (200.13 MHz): d 0.64 (3H, s) (Me-18),
1.01 (3H, s) (Me-19), 1.14 (3H, d, J¼6.1 Hz) (Me-21), 1.26
(3H, t, J¼7.1 Hz), 1.96 (1H, m, J¼2.8 Hz), 2.11 (1H, dd,
J¼2.8, 14.2 Hz), 2.59 (4H, m) (CH2, succinic), 3.94 (4H, m),
4.14 (2H, q, J¼7.1 Hz) (CH2, OEt), 4.86 (1H, dq, J¼6.1,
10.5 Hz) (H-20), 5.34 (1H, dd) (H-6); 13C NMR (50.32 MHz):
d 2.3, 14.2, 18.9, 19.9, 20.9, 24.3, 25.4, 29.2, 29.6, 31.0, 31.7,
31.7, 36.3, 36.6, 39.2, 41.8, 42.2, 49.7, 54.9, 56.1, 60.6, 64.2,
64.4, 73.3, 109.4, 122.0, 140.2, 171.6, 172.3. Anal. Calcd for
C29H44O6: C, 71.28%; H, 9.08%. Found: C, 71.46%; H, 9.20%.
References and notes
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1375, 1244, 1035, 963, 903, 883; H NMR (200.13 MHz):
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