Chiral chlorohydrins from the biocatalyzed reduction of chloroketones: Chiral building blocks for antiretroviral drugs

Article abstract of DOI:10.1002/cctc.201403023

E. coli cells that contain overexpressed alcohol dehydrogenases (ADHs) were screened as biocatalysts for the stereoselective reduction of chloroketones 5 a-d, the corresponding halohydrins 6 a-d of which are building blocks in the synthesis of antiretroviral drugs. Among them, ADH from Sphingobium yanoikuyae was found to reduce chloroketone 5 c with a high stereoselectivity (90 % de) and conversion (85 %) to furnish threo halohydrin (R,S)-6 c. ADH from Ralstonia sp. (RasADH) was able to reduce 5 a and 5 b with complementary diastereoselectivity to provide access to both threo and erythro halohydrins through "substrate-based" stereocontrol. The RasADH-catalyzed reductions were optimized to provide (R,S)-6 a with 98 % conversion and 84 % diastereomeric excess (de) and (S,S)-6 b with 95 % conversion and 86 % de. Molecular modeling studies showed that 5 b, which features a carboxybenzyl protecting group, is able to bind to the enzyme catalytic site in an "inverted" mode in comparison to tert-butyloxycarbonyl- and methyloxycarbonyl-protected substrates 5 a and 5 c, which sheds light on the observed switching of the stereopreference. RasADH-catalyzed reductions were optimized to provide (R,S)-6 a with 98 % conversion and 84 % de and (S,S)-6 b with 95 % conversion and 86 % de.

Full text of DOI:10.1002/cctc.201403023

DOI: 10.1002/cctc.201403023
Full Papers
Chiral Chlorohydrins from the Biocatalyzed Reduction of
Chloroketones: Chiral Building Blocks for Antiretroviral
Drugs
Amanda S. de Miranda,[a, b] Robert C. Simon,[c] Barbara Grischek,[c] Gabriel C. de Paula,[a]
Bruno A. C. Horta,[a] Leandro S. M. de Miranda,[a] Wolfgang Kroutil,[c] C. Oliver Kappe,*[b] and
Rodrigo O. M. A. de Souza*[a]
E. coli cells that contain overexpressed alcohol dehydrogenases
(ADHs) were screened as biocatalysts for the stereoselective re-
duction of chloroketones 5a–d, the corresponding halohydrins
6a–d of which are building blocks in the synthesis of antiretro-
viral drugs. Among them, ADH from Sphingobium yanoikuyae
was found to reduce chloroketone 5c with a high stereoselec-
tivity (90% de) and conversion (85%) to furnish threo halohy-
drin (R,S)-6c. ADH from Ralstonia sp. (RasADH) was able to
reduce 5a and 5b with complementary diastereoselectivity to
provide access to both threo and erythro halohydrins through
“substrate-based” stereocontrol. The RasADH-catalyzed reduc-
tions were optimized to provide (R,S)-6a with 98% conversion
and 84% diastereomeric excess (de) and (S,S)-6b with 95%
conversion and 86% de. Molecular modeling studies showed
that 5b, which features a carboxybenzyl protecting group, is
able to bind to the enzyme catalytic site in an “inverted” mode
in comparison to tert-butyloxycarbonyl- and methyloxycarbon-
yl-protected substrates 5a and 5c, which sheds light on the
observed switching of the stereopreference. RasADH-catalyzed
reductions were optimized to provide (R,S)-6a with 98% con-
version and 84% de and (S,S)-6b with 95% conversion and
86% de.
Introduction
More than 30 years after the identification of the human im-
munodeficiency virus (HIV), the causative agent of acquired im-
munodeficiency syndrome (AIDS), HIV infections are still one of
the world’s greatest public health challenges. It is estimated
that 35 million individuals worldwide suffer from this disease,
of which only 12.9 million have access to antiretroviral therapy,
and 2.1 million new infections and 1.5 million deaths occurred
in 2013.[1] HIV-1 protease inhibitors[2,3] are considered to be
one of the most important classes of therapeutic agents for
AIDS management and are part of the highly active antiretrovi-
ral therapy (HAART),[4] which is the first line of treatment for
AIDS. Since their introduction in the mid 1990s, HIV-1 protease
inhibitors have reduced the death rate from AIDS dramatically
in the developed world and improved clinical outcomes in
HIV-infected patients significantly by providing prolonged viral
control by suppressing viral replication. To date, 10 HIV pro-
tease inhibitors have been approved by the US Food and Drug
Administration (FDA) for clinical use, some of which, such as
saquinavir (1) and atazanavir (2), are recommended by the
World Health Organization as essential drugs for a basic health
system.[5]
HIV protease plays a crucial role in HIV replication by cleav-
ing protein precursors to generate mature, functional viral en-
zymes and structural proteins in the viral maturation process.[6]
Except for tipranavir, all FDA-approved HIV-1 protease inhibi-
tors are peptidomimetic competing inhibitors in which a scissile
bond is replaced by a noncleavable peptide bioisoster, such as
chiral hydroxyethylene or hydroxyethylamine moieties.[2]
Among them, a phenylalanine peptidomimetic moiety that
bears a chiral hydroxyethylamine group is a common structural
element of some marketed protease inhibitors, namely, 1, 2,
amprenavir (3a), fosamprenavir (3b), and darunavir (4;
Figure 1).
[a] A. S. de Miranda, G. C. de Paula, Prof. B. A. C. Horta,
Prof. L. S. M. de Miranda, Prof. R. O. M. A. de Souza
Instituto de Quꢀmica
Universidade Federal do Rio de Janeiro
Cidade Universitꢁria, 22941-909 Rio de Janeiro (Brazil)
E-mail: rodrigosouza@iq.ufrj.br
[b] A. S. de Miranda, Prof. Dr. C. O. Kappe
Institute of Chemistry
University of Graz, NAWI Graz
Heinrichstrasse 28, 8010 Graz (Austria)
E-mail: oliver.kappe@uni-graz.at
In many synthetic routes for these drugs, the introduction of
this moiety is realized by the stereoselective reduction of N-
protected chloroketones (5) as a key step to provide threo or
erythro N-protected chlorohydrins (6), followed by base treat-
ment and nucleophilic ring opening of the resulting epoxide
(7; Scheme 1).[7] Several methods for the stereoselective reduc-
tion of chloroketones 5 have been reported,[7] which include
[c] Dr. R. C. Simon, B. Grischek, Prof. Dr. W. Kroutil
Institute of Chemistry, Organic and Bioorganic Chemistry
University of Graz, NAWI Graz
Heinrichstrasse 28, 8010 Graz (Austria)
Supporting Information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201403023.
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dried E. coli cells that contain overexpressed NAD(P)H-depen-
dent alcohol dehydrogenases in a “substrate-coupled” ap-
proach using isopropanol as a cosubstrate to avoid the need
for a second enzyme for cofactor regeneration (Scheme 2).
Figure 1. Peptidomimetic HIV-1 inhibitors.
Scheme 2. Biocatalyzed reduction of 5a–d.
Results and Discussion
Freeze-dried E. coli that contain overexpressed alcohol dehy-
drogenases (ADH) from Paracoccus pantotrophus DSM 11072[38]
(PpADH), Ralstonia sp. DSM 6428[39] (RasADH), Sphingobium ya-
noikuyae DSM 6900[40] (SyADH), Lactobacillus brevis[41] (LbADH),
and Rhodococcus ruber DSM 44541[42] (RrADH-A) as well as the
wild-type organism Rhodococcus ruber DSM 44541[43] (RrADH)
were screened for the reduction of chloroketones 5a–d
(Table 1).
Scheme 1. Synthesis of chiral aminoepoxides from chloroketones. PG=Pro-
tecting group.
reduction with boron or aluminum hydrides,[8–13] Meerwein–
Ponndorf–Verley (MPV) reduction,[14] and the use of metal com-
plexes with chiral ligands.[15] These methods, however, present
many drawbacks, such as the use of expensive chiral metal
complexes, low temperatures (0 to À788C), and in some cases
the requirement of additional protection/deprotection steps or
further crystallization to achieve an acceptable diastereomeric
purity. Moreover, the majority of these methods lead to erythro
halohydrins. The preparation of diastereomerically pure threo
halohydrins is more challenging, and cost-effective reducing
methods that provide appropriate selectivity are still scarce.
The biocatalytic reduction of carbonyl groups has emerged
as an attractive alternative method for the preparation of
chiral alcohols.[16–24] Enzymes are biodegradable and renewable
catalysts that are able to catalyze reactions under environmen-
tally benign conditions, such as mild temperature, pH, and
nontoxic solvents, therefore, they are a valuable tool to fulfill
the current increasing demand for greener synthetic methods.
Most importantly, enzymes usually present high chemo-,
regio-, and stereoselectivity, so that they can shorten synthetic
routes and are especially suitable for asymmetric transforma-
tions.[17,25–30]
Table 1. Screening of biocatalysts for the reduction of chloroketones
5a–d.[a]
Entry
Biocatalyst
Substrate
de
[%][b]
Conversion
[%][b]
1
2
3
4
5
6
7
8
PpADH
PpADH
PpADH
PpADH
RasADH
RasADH
RasADH
RasADH
SyADH
SyADH
SyADH
SyADH
LbADH
LbADH
LbADH
LbADH
RrADH-A
RrADH-A
RrADH-A
RrADH-A
RrADH
5a
5b
5c
5d
5a
5b
5c
5d
5a
5b
5c
5d
5a
5b
5c
5d
5a
5b
5c
5d
5a
5b
5c
5d
82 (S,S)
88 (S,S)
38 (S,S)
–
62 (R,S)
86 (S,S)
52 (R,S)
–
78 (R,S)
32 (R,S)
90 (R,S)
–
9 (R,S)
9 (S,S)
90 (S,S)
90[c]
24
41
27
<1
42
66
40
<1
45
23
64
<1
16
37
9
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
5
–
–
–
–
<1
<1
<1
<1
11
10
21
<1
Biocatalytic methods for the reduction of N-protected chlor-
oketones such as 5 have been reported and include reductions
catalyzed by isolated engineered ketoreductases,[31–33] wild-
type,[34–36] and mutagenized micro-organisms.[37] However,
these methods possess drawbacks, such as long reaction
times, the requirement of a second enzyme for cofactor regen-
eration, or the use of biocatalysts that are not available easily
or the preparation of which requires elaborate and intricate
methods, which limits their applicability and reproducibility.
Herein we report the reduction of chloroketones 5a–d,
which bear different N-protecting groups, catalyzed by freeze-
36 (S,S)
36 (S,S)
68 (S,S)
–
RrADH
RrADH
RrADH
[a] Conditions: lyophilized cells (10 mg, 10 mgmLÀ1), potassium phos-
phate buffer (50 mm, 850 mL, 1 mm NAD(P)H, pH 7.5), substrate (10 mg,
10 mgmLÀ1) dissolved in DMSO (50 mL), isopropanol (100 mL), 700 rpm,
308C, 24 h. [b] Conversion and selectivity were determined by HPLC.
[c] Configuration of the major diastereomer could not be determined.
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Except for RrADH-A, all the biocatalysts screened reduced
chloroketones 5a–c with conversions and selectivities that
varied greatly, which depended on the N-protecting group. In
general, only low to moderate conversions were achieved,
which may be at least partially because of the low solubility of
chloroketones in the reaction media. Interestingly, although it
was the least sterically demanding substrate, 5d was a poor
substrate for all biocatalysts screened. Notably, mass transfer is
an important issue for reactions catalyzed by whole-cell biocat-
alysts, and therefore, the physicochemical properties of the
substrate, which account for its solubility and ability to cross
membranes, can also play a role in the biocatalyst per-
formance.[44]
the re face and the remaining 67% would react on the si face.
Substrate 5a is able to bind to the enzyme in a way that the
phenylalanine ring is oriented towards helix a6 and buried in
a pocket formed by residues Ala182, Ile188, Leu201, Phe205,
and Val214, whereas the N-protecting group (Boc) is oriented
towards helix a5 and is located in a second pocket formed
mainly by residues Ser137, Val 138, Ala139, Leu144, His147,
and Phe205 (Figure 2a). Phe205 is present in both pockets and
PpADH was able to reduce chloroketones 5a–c and dis-
played an anti-Prelog selectivity (for chlorohydrins 6a–d, the
largest group has the lowest Cahn–Ingold–Prelog (CIP) priori-
ty), which gave (S,S)-halohydrins with a higher diastereomeric
excesses (de) for substrates with tert-butyloxycarbonyl (Boc;
5a) and carboxybenzyl (Cbz; 5b) protecting groups (entries 1
and 2, Table 1) than for the smaller methyloxycarbonyl (Moc)
group (5c; entry 3, Table 1). Anti-Prelog selectivity has been re-
ported for the reduction of ketones by cells of P. pantotro-
phus,[45] whereas the biocatalyst used here, E. coli whole cells
that contain a short-chain alcohol dehydrogenase of P. panto-
trophus,[38] was reported previously to reduce ketones with
Prelog selectivity, which includes a-chloroacetophenone. None-
theless, these data were obtained mainly from the reduction of
aryl alkyl ketones, which have a low structural similarity with
the substrates used in this work.
Alcohol dehydrogenases from Ralstonia sp. and Sphingobium
yanoikuyae have been reported to reduce “small-bulky” ke-
tones (ketones that bear one small and one large substituent)
as well as “bulky-bulky” aryl alkyl ketones (ketones in which
the carbonyl group is adjacent to two bulky groups), which ex-
hibits Prelog selectivity in both cases.[39,46,47] The reduction of
5a–c catalyzed by SyADH furnished (R,S)-chlorohydrins with
similar selectivity and conversion for 5a and 5c (entries 9 and
11), which bear aliphatic protecting groups, and only poor se-
lectivity and conversion for 5b. Prelog selectivity was also ob-
served in the RasADH-catalyzed reduction of 5a and 5c (en-
tries 5 and 7), whereas a distinct behavior was observed for 5b
(entry 6), which was reduced with the opposite selectivity to
give diastereomerically complementary halohydrin (S,S)-6b.
To better understand the rationale behind the switch of ster-
eopreference in the catalytic reduction promoted by ADH from
Ralstonia sp. towards substrate 5b, a molecular docking study
of 5a–c in the catalytic site of this enzyme was performed
using the Dockthor program.[48–50] The configurations obtained
after 30 docking runs per ligand with 3 million evaluations per
run were subjected to statistical analysis based not only on the
energy but also considering the maximum distance of 4.5 ꢁ
between the C4N carbon atom of the nicotinamide ring and
the carbon atom of the carbonyl group of the substrate. As
a result, for 5a and 5c 83 and 80% of the configurations, re-
spectively, were oriented in such a way that the hydride trans-
fer would occur on the re face. In contrast, for substrate 5b,
only 33% of the configurations would lead to a reaction on
Figure 2. Docking of compounds a) 5a, b) 5c, and c) 5b in the catalytic site
of ADH from Ralstonia sp. and d) the superposition of docked structures.
Compounds 5a, 5b, and 5c are shown in red, blue, and green, respectively,
and protecting groups are shown as ball and stick representations. (Recep-
tor coordinates were extracted from the enzyme indexed in the Protein Da-
tabase (PDB) under code 4BMS).[46]
splits the active site into two regions. The presence of Phe205
in this position imposes several restrictions to the binding of
bulky substrates. In the case of 5c (Figure 2b), the small size
of the Moc protecting group allows a number of possible bind-
ing modes (Figure S31). Interestingly, the binding mode is
completely different if we compare Boc- and Moc-protected
5a and 5c to 5b, which features a Cbz group. In the latter
case, the substrate molecule is “inverted”, the phenyl group of
the Cbz moiety occupies the first binding pocket (near to helix
a6), and the phenylalanine ring occupies the second pocket
(Figure 2c). This is the opposite of that observed in the case of
5a and 5c (Figure 2d). In this way of binding, the a-chlorocar-
bonyl group of the substrate is oriented so that hydride trans-
fer occurs at the si face, which supports the fact that substrate
5b is reduced by RasADH to give the erythro diastereomer as
the major product, that is, chlorohydrin (S,S)-6b.
The switch of stereopreference exhibited by RasADH-cata-
lyzed reductions towards substrates with different N-protecting
groups was advantageous as it could allow a “substrate-based”
stereocontrol approach[51–58] to give both threo and erythro hal-
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ohydrins. The inversion of the stereochemical outcome of an
enzymatic reaction is still challenging, although it may be ac-
complished through enzyme, substrate, and medium engineer-
ing.[54] Access to both product stereoisomers from the same
substrate can be obtained mainly by using two stereocomple-
mentary enzymes by “enzyme-based” stereocontrol, or alterna-
tively, by the use of a single enzyme that displays complemen-
tary stereoselectivity towards two structurally related sub-
strates, named “substrate-based” stereocontrol. The latter ap-
proach has been reported for the reduction of the C=C bond
of a,b-unsaturated ketones catalyzed by enoate reductases
from the Old Yellow Enzyme family, the stereopreference of
which could be switched by subtle changes in the substitu-
ents, protecting group, or ring size of the substrate as well as
by switching the relative configuration of the C=C
bond.[51–54,57,58] This strategy was employed successfully in the
bioreduction of b-cyanoacrylates catalyzed by ene reductase to
provide access to both enantiomers of chiral pregabalin pre-
cursors.[57] For the reduction of chloroketones 5, single keteore-
ductases with complementary stereoselectivity for substrates
that bear different protecting groups were reported during
biocatalyst screening,[31,32] but the reactions were not further
optimized to develop a process that is able to furnish both dia-
stereomers with acceptable conversions and diastereomeric
excess through reduction catalyzed by a single biocatalyst.
LbADH[41] and RrADH-A[42,59,60] have been reported to reduce
a broad range of ketones, which include a-chloroketones.
However, they were not suitable for the reduction of sub-
strates 5a–d (entries 13–20). As the reduction of ketones by
whole cells of Rhodococcus ruber DSM 44541 was reported to
occur by means of other ADHs than RrADH-A,[47] an attempt to
perform the reaction with this biocatalyst was also undertaken,
but only poor conversions were achieved (entries 21–24).
Table 2. Reduction of chloroketones 5a–c catalyzed by PpADH.[a]
Entry
Substrate
de
[%][b]
Conversion
[%][b]
Isopropanol
[%]
DMSO
[%]
1
2
3
4
5
6
7
8
9
10
5b
5b
5b
5a
5c
5b
5b
5b
5a
5c
82 (S,S)
92 (S,S)
88 (S,S)
92 (S,S)
84 (S,S)
74 (S,S)
90 (S,S)
90 (S,S)
90 (S,S)
56 (S,S)
44
40
22
32
29
31
80
43
58
57
20
50
70
50
50
5
5
5
5
5
5
5
5
5
5
20
50
60
50
50
[a] Conditions: lyophilized cells (10 mg, 10 mgmLÀ1), potassium phos-
phate buffer (50 mm, 250–750 mL, 1 mm NADH, pH 7.5), substrate (10 mg,
10 mgmLÀ1) dissolved in DMSO (50–600 mL), isopropanol (50–700 mL),
700 rpm, 308C, 24 h. [b] Conversion and selectivity were determined by
HPLC.
screening conditions, we performed the reaction with increas-
ing isopropanol and DMSO concentrations (Table 2).
The reduction of 5b could be performed at isopropanol con-
centrations up to 50% without any significant decrease in con-
version (entry 2), whereas a concentration as high as 70% was
found to be detrimental (entry 3). It is possible that isopropa-
nol decreases biocatalyst activity and increases substrate solu-
bility simultaneously, which results in opposite effects on con-
version. If the reaction is performed with 50% isopropanol, the
reduction of 5c occurred with a higher stereoselectivity
(entry 5). The increase of the DMSO concentration to 50% im-
proved the conversion of 5b greatly, so that chlorohydrin 6b
was obtained with 80% conversion and an excellent diastereo-
meric excess (entry 7). Such an effect was described previously
for ketone reductions catalyzed by PpADH and seems to be re-
lated to the stabilization of the enzyme by DMSO instead of
activation.[38] Under the same conditions, the reductions of 5a
and 5c were also improved, although only moderate conver-
sions could be achieved (entries 9 and 10).
Reaction optimization
The use of organic solvents in whole-cell biocatalyzed reac-
tions, although limited by biocatalyst stability, offers some ad-
vantages such as the improvement of substrate solubility in
the reaction medium, which allows higher substrate loadings,
easier recovery of product from the aqueous medium, and the
reduction of toxic substrates and products around enzymes if
biphasic organic solvent/buffer systems are employed. In addi-
tion, reaction stereoselectivity can be improved by the use of
organic solvents through the inhibition of competing enzymes
as well as through a direct effect on the target enzyme.[44,61–63]
The optimization of reactions that furnished promising re-
sults for at least one substrate was performed. In most cases,
optimization relied on attempts to increase the amount of or-
ganic solvent in the reaction medium and was driven by avail-
able literature data for each biocatalyst.
The optimization of SyADH-catalyzed reductions by chang-
ing the isopropanol concentration in the reaction medium was
also attempted (Table 3). For the SyADH-catalyzed reduction of
5a, an increase of the concentration of isopropanol from 10 to
20% resulted in a lower conversion (entry 3). However, a de-
crease of the isopropanol concentration to 5% did not affect
the reduction of 5a but resulted in improved conversion for
5b and 5c (entries 5 and 8). Under these conditions, halohy-
drin (R,S)-5c was obtained with a good conversion (85%) and
in an excellent diastereomeric ratio (90%).
Ethanol is not used commonly in biocatalytic reductions as
enzymes can be poisoned by the coproduct acetaldehyde pro-
duced during the reaction. Nevertheless, some examples of
the use of ethanol as a cosubstrate in a “substrate-coupled ap-
proach” can be found in the literature, which includes the re-
duction of bulky-bulky ketones catalyzed by Sphingobium ya-
noikuyae and Ralstonia sp. cells.[47] We took this into account
and attempted to optimize SyADH-catalyzed reductions using
ethanol instead of isopropanol as the cosubstrate. However,
PpADH has been reported to be highly tolerant to organic
solvents, especially DMSO,[38] which may be related to the abili-
ty of P. pantotrophus to grow on media with a high sulfur con-
tent. To optimize PpADH-catalyzed reductions, which furnished
chlorohydrin 6b with a promising diastereomeric excess under
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and excellent diastereomeric excess (84%), whereas the best
compromise between conversion and selectivity for the reduc-
tion of 5b was achieved with 15% isopropanol to provide hal-
ohydrin (S,S)-6b with 82% conversion and a diastereomeric
excess of 80% (Figure 3b).
Table 3. Reduction of chloroketones 5a–c catalyzed by SyADH.[a]
Entry
Substrate
de
[%][b]
Conversion
[%][b]
Isopropanol
[%]
EtOH
[%]
1
2
3
4
5
6
7
8
5a
5a
5a
5a
5b
5b
5b
5c
5c
5c
5c
87 (R,S)
78 (R,S)
74 (R,S)
80 (R,S)
29 (R,S)
32 (R,S)
64 (R,S)
90 (R,S)
90 (R,S)
92 (R,S)
88 (R,S)
50
45
25
13
31
23
8
85
64
27
59
5
10
20
–
5
10
–
5
10
–
–
–
–
5
–
–
5
–
–
5
–
Further optimization of the reduction of 5a,b was attempt-
ed by using ethanol instead of isopropanol as the cosubstrate
as Ralstonia sp. cells have been reported to be able to use
both ethanol and isopropanol as a hydrogen source and to
present better apparent activity if ethanol was used instead of
isopropanol in the reduction of bulky-bulky ketones.[47] In addi-
tion, this biocatalyst was reported to be more active at pH 7.5
if ethanol was used, whereas the best pH for reactions with
isopropanol was 7.0. We took this into account and performed
some experiments at pH 7.0. The results are summarized in
Table 4.
9
10
11
5
[a] Conditions: lyophilized cells (10 mg, 10 mgmLÀ1), potassium phos-
phate buffer (50 mm, 750–900 mL, 1 mm NADPH, pH 7.5), substrate
(10 mg, 10 mgmLÀ1) dissolved in DMSO (50 mL), isopropanol (50–200 mL)
or ethanol (50 mL), 700 rpm, 308C, 24 h. [b] Conversion and selectivity
were determined by HPLC.
Table 4. Reduction of chloroketones 5 catalyzed by RasADH.[a]
Entry
Substrate
de
[%][b]
Conversion
[%][b]
Isopropanol
[%]
EtOH
[%]
this strategy was not successful and led to low conversions for
all substrates (entries 4, 7, and 10).
1
5a
5a
5a
5a
5a
5a
5b
5b
5b
5b
5b
5c
5d
84 (R,S)
84 (R,S)
53 (R,S)
74 (R,S)
82 (R,S)
90 (R,S)
82 (S,S)
76 (S,S)
82 (S,S)
80 (S,S)
73 (S,S)
16 (R,S)
14[d]
98
85
35
49
83
72
80
66
51
75
75
41
23
40
40
–
–
–
–
–
5
20
40
50
–
–
5
10
20
–
2[c]
3
The effect of isopropanol concentration on RasADH-cata-
lyzed reductions was assessed for 5a and 5b (Figure 3), which
are reduced by this biocatalyst with opposite selectivities to
lead to both threo and erythro chlorohydrins. An optimum iso-
propanol concentration of 40% was found for the reduction of
5a (Figure 3a), which gave (R,S)-6a with almost full conversion
4
5
6
7
8[c]
9
10
11
12
13
–
15
15
–
–
–
40
40
–
[a] Conditions: lyophilized cells (10 mg, 10 mgmLÀ1), potassium phos-
phate buffer (50 mm, 550–900 mL, 1 mm NADPH, pH 7.5), substrate
(10 mg, 10 mgmLÀ1) dissolved in DMSO (50 mL), isopropanol (150-400 mL)
or ethanol (50-500 mL), 700 rpm, 308C, 24 h. [b] Conversion and selectivity
were determined by HPLC. [c] Reactions performed at pH 7.0. [d] Configu-
ration of the major diastereomer could not be determined.
For both 5a and 5b, the change of the pH from 7.5 to 7.0
did not affect stereoselectivity but decreased conversions (en-
tries 2 and 8). If ethanol was used instead of isopropanol as
the cosubstrate, the stereoselectivity was improved slightly al-
though conversions were lower. Notably, the reduction of 5a
with ethanol gave the threo halohydrin 6b with the highest
diastereomeric excess (entry 6).
The reduction of 5c and 5d by RasADH was also performed
with 40% isopropanol at pH 7.5 as these were the best condi-
tions with regard to selectivity and conversion. Under these
conditions, threo halohydrin 6c was obtained with a similar
conversion (41%) but a lower diastereomeric excess (16%;
entry 12) in comparison to those achieved under the screening
conditions (entry 7, Table 1), whereas the reduction of 5d
could be achieved (entry 13), although with a low conversion
(23%) and selectivity (14% de). Under the screening conditions
(entries 4, 8, 12, 16, and 24, Table 1), none of the biocatalysts
could reduce 5d with a conversion higher than 5%. This in-
Figure 3. Effect of isopropanol on the reduction of a) 5a and b) 5b cata-
lyzed by RasADH. Conditions: lyophilized cells (10 mg, 10 mgmLÀ1), potassi-
um phosphate buffer (50 mm, 50–900 mL, 1 mm NADPH, pH 7.5), substrate
(10 mg, 10 mgmLÀ1) dissolved in DMSO (50 mL), isopropanol (50–900 mL),
700 rpm, 308C, 24 h. Conversion and selectivity were determined by HPLC.
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crease in conversion that results from an increase in isopropa-
nol concentration is likely to be at least partially related to an
improvement in substrate solubility.
The effect of other organic solvents on the RasADH-cata-
lyzed reduction of 5a and 5b was evaluated by performing
the reaction using the optimum isopropanol concentration for
each substrate and 20% v/v of an organic cosolvent (Figure 4).
Figure 5. Effect of organic cosolvent on the reduction of 5a catalyzed by
RasADH at an isopropanol concentration of 15% v/v. Conditions: lyophilized
cells (10 mg, 10 mgmLÀ1), potassium phosphate buffer (50 mm, 600 mL,
1 mm NADPH, pH 7.5), substrate (10 mg, 10 mgmLÀ1) dissolved in DMSO
(50 mL), isopropanol (150 mL), organic cosolvent (200 mL), 700 rpm, 308C,
24 h. Conversion and selectivity were determined by HPLC.
duction of 5a was also performed with only 15% isopropanol
and 20% organic cosolvent and similar or better conversions
were achieved, although the selectivity decreased (Figure 5).
The effect of substrate concentration on the RasADH-cata-
lyzed reduction of 5a under optimum conditions was also as-
sessed (Figure 6). However, the substrate concentration could
Figure 4. Effect of organic cosolvent (20%) on the reduction of a) 5a and
b) 5b catalyzed by RasADH isopropanol concentrations of 40 and 15% v/v,
respectively. Conditions: a) lyophilized cells (10 mg, 10 mgmLÀ1), potassium
phosphate buffer (50 mm, 350 mL, 1 mm NADPH, pH 7.5), substrate (10 mg,
10 mgmLÀ1) dissolved in DMSO (50 mL), isopropanol (400 mL), organic cosol-
vent (200 mL), 700 rpm, 308C, 24 h; b) lyophilized cells (10 mg, 10 mgmLÀ1),
potassium phosphate buffer (50 mm, 600 mL, 1 mm NADPH, pH 7.5), sub-
strate (10 mg, 10 mgmLÀ1) dissolved in DMSO (50 mL), isopropanol (150 mL),
organic cosolvent (200 mL), 700 rpm, 308C, 24 h. Conversion and selectivity
were determined by HPLC.
Figure 6. Effect of substrate concentration on the reduction of 5a under op-
timized conditions. Conditions: lyophilized cells (10 mgmLÀ1), potassium
phosphate buffer (50 mm, 550 mL, 1 mm NADPH, pH 7.5), substrate (10–
50 mg, 10–50 mgmLÀ1) dissolved in DMSO (50 mL), isopropanol (400 mL),
700 rpm, 308C, 24 h. Conversion and selectivity were determined by HPLC.
Both water-immiscible (tert-butyl methyl ether (TBME), toluene,
diisopropyl ether (DIPE), heptane) and water-miscible solvents
(THF, DMSO) were used.
not be increased without a striking decrease in conversion,
which was reduced from 98 to 31% if the substrate loading
was doubled.
For the reduction of 5a, a decrease in conversion was ob-
served in all cases, whereas selectivity was unaffected or slight-
ly improved (Figure 4a). However, the reduction of 5b was
also optimized by the addition of toluene, DIPE, or DMSO to
give chlorohydrin (S,S)-6b with a high diastereomeric excess
(86%) and almost full conversion (95%) if DMSO was used (Fig-
ure 4b). The reductions of 5a were performed with 40% iso-
propanol so that the total organic solvent content (60%) was
higher than that employed in the reduction of 5b (35%). Nev-
ertheless, 5a was still reduced with moderate conversions in
such a high organic-solvent-content reaction medium if DIPE,
heptane, or DMSO were used, which shows the high tolerance
of the biocatalyst to organic solvents. For comparison, the re-
Finally, reactions were performed under the optimized con-
ditions on a 50 mg scale to provide both threo and erythro
chlorohydrins (Table 5). Products were isolated and purified to
give chiral chlorohydrins 5a–c in good yields (70–84%) and
high diastereomeric excesses (84–90%). In general, conversions
were slightly higher in comparison to that obtained on
a 10 mg scale. For the reduction of 5a catalyzed by RasADH
with 40% v/v isopropanol, a conversion of 97% could be ach-
ieved after 12 h.
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5 mm) and an UV detector, using acetonitrile, water, and 0.1% tri-
fluoroacetic acid as the mobile phase. Analyses were performed at
378C using mobile phase A (water/acetonitrile 90:10 v/v +0.1%
TFA) and a mobile phase B (acetonitrile+0.1% TFA) at a flow rate
of 1.5 mLminÀ1. For the HPLC analysis of 5a–b and 6a–b the fol-
lowing gradient was applied: linear increase from 30% B to 100%
B in 8 min, hold at 100% B for 2 min. For HPLC analysis of 5c–d
and 6c–d the following gradient was applied: linear increase from
15% B to 100% B in 8 min, hold at 100% B for 2 min. Compound-
s 6a–c obtained from biocatalytic reactions were characterized by
NMR spectroscopy, UV spectroscopy, optical rotation, and HRMS
(TOF-MS EI+).
Table 5. Biocatalyzed reduction of chloroketones 5a–c on a 50 mg scale.
Entry
Biocatalyst
Substrate
de
[%][a]
Conversion
[%][a]
Yield
[%]
1[b]
2[c]
3[d]
4[e]
RasADH
RasADH
RasADH
SyADH
5a
5a
5b
5c
84 (R,S)
90 (R,S)
86 (S,S)
90 (R,S)
99
81
98
97
82
70
80
84
[a] Conversion and stereoselectivity were determined by HPLC. [b] Reac-
tion performed in phosphate buffer (pH 7.5) that contained 40% v/v iso-
propanol [c] Reaction performed in phosphate buffer (pH 7.5) that con-
tained 50% v/v ethanol. [d] Reaction performed in phosphate buffer
(pH 7.5) that contained 15% isopropanol and 20% DMSO. [e] Reaction
performed in phosphate buffer (pH 7.5) that contained 5% isopropanol.
Preparation of biocatalysts
The various ADHs were overexpressed heterologously in E. coli and
freeze dried to give the ready-to-use whole-cell preparation as de-
scribed previously using the following plasmids (pEG refers to the
internal plasmid number): pEG51 for the RasADH from Ralstonia
sp.,[39] pEG16 for PpADH from Paracoccus pantotrophus,[38] pEG53
for SyADH from Sphingobium yanoikuyae,[40] pEG180 for LbADH
from Lactobacillus brevis,[41] and pEG10: RrADH-A from Rhodococcus
ruber.[42] For optimal expression, the following salts were added to
the growth medium depending on the ADH: RasADH; 0.6 mm
CaCl2; RrADH-A: 100 mgLÀ1 ZnCl2. Freeze-dried cells of Rhodococ-
cus ruber DSM 44541 (RrADH) were prepared as described previ-
ously.[43]
Conclusions
We employed lyophilized E. coli cells that contained overex-
pressed alcohol dehydrogenases to obtain threo and erythro
chlorohydrins 6 with different N-protecting groups with high
conversions and diastereomeric purity comparable to that ob-
tained by reduction methods published previously. These com-
pounds are key building blocks for the synthesis of marketed
antiretroviral drugs, such as atazanavir, darunavir, and ampre-
navir, and are also likely to be useful for medicinal chemistry
purposes as they feature a pharmacophore encountered fre-
quently in HIV-1 protease inhibitors. The transformations de-
scribed herein were performed under mild, environmentally
benign conditions using isopropanol as the cosolvent and hy-
drogen source in a substrate-coupled approach, which avoided
the use of enzymes for cofactor recycling. Notably, the alcohol
dehydrogenase from Ralstonia sp. provided access to both dia-
stereomers of chlorohydrin 6 by changing the protecting
group of the substrate, so that a substrate-based stereocontrol
was enabled as a strategy to obtain stereocomplementary
chiral chlorohydrins (R,S)-6a and (S,S)-6b, both of which are
useful as building blocks for antiretroviral drugs. Additionally,
this biocatalyst was also highly tolerant to organic solvents,
which allowed reactions to be performed under conditions in
which substrates were fully soluble in the reaction medium to
give the product with high conversions.
General procedure for bioreduction
Lyophilized cells of E. coli that contained overexpressed ADHs
(10 mg) were rehydrated in potassium phosphate buffer (850 mL,
50 mm, pH 7.5, NAD(P)H 1 mm) for 30 min at 308C and 700 rpm on
a rotatory shaker in a 1.5 mL vial. Then, chloroketone 5 (10 mg,
0.15–0.19 mmol) dissolved in DMSO (50 mL) was added followed by
isopropanol (100 mL). Reactions were shaken for 24 h at 308C and
700 rpm on a rotatory shaker, which was operated horizontally.
Then, reactions were quenched by extraction with ethyl acetate
(2ꢂ1.5 mL). The organic phase was separated from the cells by
centrifugation, dried on MgSO4, and analyzed by HPLC. Diastereo-
mer assignment was performed by comparison of HPLC chromato-
grams from the reaction with chromatograms of diastereoisomeric
mixtures of 6a–c obtained from the non-stereoselective reduction
of 5a–c with NaBH4 at RT, and chromatograms of diastereomerical-
ly pure 6a–c obtained according to published diastereoselective
methods, the diastereoselectivity of which was known (Table S1).
Experimental Section
Reduction of 5a catalyzed by RasADH (50 mg scale)
General
Lyophilized cells of E. coli that contained recombinant ADH from
Ralstonia sp. DSM 6428 (50 mg, 0.17 mmol) were rehydrated in po-
tassium phosphate buffer (2750 mL, 50 mm, pH 7.5, NADPH 1 mm)
for 30 min at 308C and 700 rpm on a rotatory shaker in a 10 mL
vial. Then, isopropanol (2000 mL) followed by chloroketone 5a
(50 mg) dissolved in DMSO (250 mL) were added, and the reaction
was shaken for 24 h at 308C and 700 rpm on a rotatory shaker,
which was operated horizontally. Extraction with ethyl acetate (4ꢂ
50 mL) was performed, and the organic phase was dried over
MgSO4 and analyzed by HPLC. The organic phase was evaporated,
and the product was purified by flash chromatography to give
chlorohydrin (R,S)-6a (42 mg, 0.14 mmol) in 82% yield. 1H NMR
(300 MHz, CDCl3, 258C, TMS): d=1.43 (s, 9H; CH3), 2.92 (dd,
Solvents and chemicals were purchased from standard commercial
sources and used as received unless otherwise stated. Chloroke-
tones 5a–c were synthesized according to the method of Pinho
et al.,[64] and chloroketone 5d was prepared from 5b by methods
reported previously.[65,66] 1H and 13C{1H} NMR spectra were recorded
by using a 300 MHz Bruker instrument at 300 and 75 MHz, respec-
tively. Chemical shifts are expressed in ppm downfield from tetra-
methylsilane (TMS) as the internal standard. Signal multiplicities
are indicated by the letters s (singlet), d (doublet), dd (doublet of
doublets), t (triplet), and m (multiplet). HPLC analysis was per-
formed by using a Shimadzu LC20 chromatograph equipped with
a C18 reversed-phase analytical column (150ꢂ4.6 mm, particle size
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J(H,H)=9 Hz, J(HH)=13.5, 1H, CH2), 3.01 (dd, J(H,H)=9 Hz, J(HH)=
13.5, 1H, CH2), 3.17 (bs, 1H, OH), 3.56 (m, 2H; CH2), 3.74 (m, 1H;
CH), 3.86 (dd, J(HH)=3 Hz, J(HH)=9 Hz, 1H, CHOH), 4.91(d,
J(H,H)=9 Hz,1H; NH), 7.22–7.35 ppm (m, 5H; ArH); 13C NMR
(50 MHz,CDCl3, 258C): d=28.29, 38.31, 47.83, 53.97, 71.48, 79.95,
126.60, 128.59, 129.30, 137.84, 156.20 ppm; IR (KBr): n˜ =3444, 3373,
3031, 2983, 2968, 2930, 1668, 1525, 1164 cmÀ1; [a]D20 =À36 (c=0.07
in CHCl3); HRMS (TOF-MS EI+): m/z: calcd for C10H14ClNO3:
199.076391 [MÀ(CH3)3COCO]+; found: 199.076012.
were performed at 378C using mobile phase A (water/acetonitrile
90:10 v/v + 0.1% TFA) and mobile phase B (acetonitrile+0.1%
TFA) at a flow rate of 1.5 mLminÀ1. For the analysis of 5a–b and
6a–b the following gradient was applied: linear increase of solu-
tion B from 30 to 100% B in 8 min, hold at 100% B for 2 min. For
the analysis of 5c–d and 6c–d the following gradient was applied:
linear increase of solution B from 15 to 100% B in 8 min, hold at
100% B for 2 min. For comparison, chloroketones 5a–d were re-
duced with NaBH4 in THF at RT to give diastereomeric mixtures of
chlorohydrins 6a–d. Chloroketones 5a–c were also reduced sterer-
eoselectively by methods reported previously to give (S,S)-6a,[9]
(R,S)-6a,[11] (S,S)-6b,[12] (R,S)-6b,[11] and (S,S)-6c.[67] The diastereomers
of 6a–c obtained by biocatalytic reactions were assigned by com-
parison of chromatograms from diastereomeric mixtures and dia-
stereomerically enriched chlorohydrins with chromatograms ob-
tained from the reactions. As there is no reported method for the
stereoselective reduction of 5d, the diastereomers of 6d could not
be assigned. The diastereomeric excess for each reaction was cal-
culated by the difference of the relative area of peaks assigned to
each diastereomer.
Reduction of 5b catalyzed by RasADH (50 mg scale)
Lyophilized cells of E. coli that contained recombinant ADH from
Ralstonia sp. DSM 6428 (50 mg, 0.15 mmol) were rehydrated in po-
tassium phosphate buffer (3250 mL, 50 mm, pH 7.5, NADPH 1 mm)
for 30 min at 308C and 700 rpm on a rotatory shaker in a 10 mL
vial. Isopropanol (750 mL) followed by chloroketone 5b (50 mg) dis-
solved in DMSO (1000 mL) were added, and the reaction was
shaken for 24 h at 308C and 700 rpm on a rotatory shaker, which
was operated horizontally. Extraction with ethyl acetate (4ꢂ50 mL)
was performed, and the organic phase was dried over MgSO4 and
analyzed by HPLC. The organic phase was evaporated, and the
product was purified by flash chromatography to give chlorohydrin Acknowledgements
1
(S,S)-6a (40 mg, 0.12 mmol) in 80% yield. H NMR (300 MHz, CDCl3,
258C, TMS): d=2.92–3.05 (m, 3H; CH2,CHOH), 3.61 (dd, J(H,H)=
C.O.K acknowledges the Science without Borders program (CNPq,
3 Hz, J(HH)=12, 1H, CH2), 3.68 (dd, J(H,H)=3 Hz, J(HH)=12, 1H,
CH2), 3.88 (bs, 1H, OH), 3.99–4.01 (m, 1H; CH), 4.84(bd, J(H,H)=
6 Hz,1H; NH), 5.06 (s, 2H; CH2Ar), 7.20–7.40 ppm (m, 10H; ArH);
13C NMR (50 MHz, CDCl3, 258C): d=35.64, 47.60, 54.69, 66.97, 73.21,
126.76, 127.99, 128.21, 128.54, 128.65, 129.44, 136.38, 137.01,
156.17 ppm; IR (KBr): n˜ =3320, 3061, 3029, 2952, 1684, 1541, 1259,
1026 cmÀ1; [a]2D0 =À24.7 (c=0.07 in MeOH); HRMS (TOF-MS EI+):
m/z: calcd for C18H20ClNO3: 333.113171 [M]+; found: 333.114365.
CAPES) for a “Special Visiting Researcher” fellowship. A.S.M. ac-
knowledges the Science without Borders program (CNPq) for
a doctoral scholarship (SWE 221255/2013-3). We thank Vagner D.
Pinho for the synthesis of compounds 5a–c.
Keywords: biocatalysis
·
diastereoselectivity
·
enzyme
catalysis · ketones · reduction
Reduction of 5c catalyzed by SyADH (50 mg scale)
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Conversion and diastereomeric excess were determined by HPLC
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4.6 mm, particle size 5 mm) with a UV detector (215 nm). Analyses
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Received: December 15, 2014
Published online on && &&, 0000
ChemCatChem 0000, 00, 0 – 0
www.chemcatchem.org
9
ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

FULL PAPERS
A. S. de Miranda, R. C. Simon, B. Grischek,
G. C. de Paula, B. A. C. Horta,
Choose your side, not your enzyme: A
single alcohol dehydrogenase is able to
reduce structurally related chloroke-
tones derived from S-amino acids with
the opposite enantiopreference to fur-
nish both (R,S) and (S,S)-chlorohydrins.
Stereocontrol is achieved by changing
the N-protecting group in the substrate,
which allows the preparation of stereo-
complementary halohydrins using the
same biocatalyst.
L. S. M. de Miranda, W. Kroutil,
C. O. Kappe,* R. O. M. A. de Souza*
&& – &&
Chiral Chlorohydrins from the
Biocatalyzed Reduction of
Chloroketones: Chiral Building Blocks
for Antiretroviral Drugs
&
ChemCatChem 0000, 00, 0 – 0
www.chemcatchem.org
10
ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!