Deep Eutectic Solvents as Media in Alcohol Dehydrogenase-Catalyzed Reductions of Halogenated Ketones

Article abstract of DOI:10.1002/cctc.201901582

The application of deep eutectic solvents (DESs) in biotechnological processes has gained an outstanding relevance, as they can be used as greener media to obtain higher productivities and selectivities. In the present contribution, an eutectic mixture composed of choline chloride (ChCl): glycerol (1 : 2 mol/mol) has been used as a reaction medium in combination with Tris?SO₄ 50 mM buffer pH 7.5, applied to the alcohol dehydrogenase (ADH)-catalyzed reduction of various carbonyl precursors of chiral halohydrins. These alcohols are key intermediates of biologically active compounds, and hence they are of industrial interest. In the presence of up to 50 % v/v of DES, these biotransformations were achieved up to 300–400 mM of the α-halogenated ketone substrate, getting access to the final compounds with excellent conversions (usually >90 %) and enantiomeric excess (ee >99 %). Among the different ADHs tested, two stereocomplementary enzymes (Lactobacillus brevis ADH and Rhodococcus ruber ADH) afforded the best results, so both alcohol enantiomers could be obtained in all the studied examples. Selected bioreductions were scaled up to 250 mg and 1 g, demonstrating the potential that DESs can offer as media in redox processes for substrates with low solubility in water.

Full text of DOI:10.1002/cctc.201901582

Accepted Article
Title: Deep Eutectic Solvents as Media in Alcohol Dehydrogenase-
Catalyzed Reductions of Halogenated Ketones
Authors: Fatima Zohra Ibn Majdoub Hassani, Saaid Amzazi, Joseph
Kreit, and Ivan Lavandera
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Deep Eutectic Solvents as Media in Alcohol Dehydrogenase-
Catalyzed Reductions of Halogenated Ketones
Fatima Zohra Ibn Majdoub Hassani,[a,b] Saaid Amzazi, Joseph Kreit, and Iván Lavandera*
[a]
[a]
[b]
Abstract: The application of deep eutectic solvents (DESs) in
biotechnological processes has gained an outstanding relevance, as
they can be used as greener media to obtain higher productivities and
selectivities. In the present contribution, a eutectic mixture composed
of choline chloride (ChCl): glycerol (1:2 mol/mol) has been used as a
is the reduction of the α-halogenated ketone precursors. Hence,
the use of metals as catalysts has provided appreciable results,[
4]
but from an environmental point of view and selectivity, the
utilization of enzymes is more desirable. In fact, in the recent
literature, many examples can be found employing alcohol
dehydrogenases (ADHs),[5] in order to obtain chiral halohydrins.
These NAD(P)-dependent oxidoreductases have provided
impressive results in terms of activity and selectivity under mild
conditions. Thus, whole cells[ and isolated or overexpressed
enzymes[7] have been utilized for this purpose.
4
reaction medium in combination with Tris-SO 50 mM buffer pH 7.5,
applied to the alcohol dehydrogenase (ADH)-catalyzed reduction of
various carbonyl precursors of chiral halohydrins. These alcohols are
key intermediates of biologically active compounds, and hence they
are of industrial interest. In the presence of up to 50% v/v of DES,
these biotransformations were achieved up to 300-400 mM of the α-
halogenated ketone substrate, getting access to the final compounds
with excellent conversions (usually >90%) and enantiomeric excess
6]
(ee >99%). Among the different ADHs tested, two
stereocomplementary enzymes (Lactobacillus brevis ADH and
Rhodococcus ruber ADH) afforded the best results, so both alcohol
enantiomers could be obtained in all the studied examples. Selected
bioreductions were scaled up to 250 mg and 1 g, demonstrating the
potential that DESs can offer as media in redox processes for
substrates with low solubility in water.
Figure 1. Examples of active ingredients of commercialized medicaments
where halohydrins are key intermediates (the part of the molecule derived from
the halohydrin appears in red).
Introduction
In the last years, the synthesis of chiral halohydrins has received
great attention due to their synthetic versatility and high reactivity.
They have become privileged scaffolds to access biologically
active derivatives. They are precursors of β-adrenoreceptor
blockers[1] and also key intermediates of various active
ingredients of medicaments that are currently commercialized.
Examples of these compounds (Figure 1) include mirabegron[2]
and ezetimibe,[3] used for treatment of overactive bladder and
hypercholesterolemia, respectively.
Aqueous medium is the natural environment for these
biocatalysts, but it has been discovered that they can work in non-
conventional media containing organic solvents[8] or ionic liquids
ILs).[9] This can be advantageous for these enzymes due to the
(
large hydrophobicity of the organic substrates, making feasible
the employment of higher substrate concentrations and therefore,
higher productivities. Related to this fact, new types of ionic liquids
that are ecologically more convenient than the organic solvents,
are the (natural) deep eutectic solvents (NA)DESs.[10] They are
Among the plethora of synthetic methods to synthesize
halohydrins in enantioenriched form, probably the most employed
typically made of a hydrogen bond donor (HBD, such as glycerol,
ethylene glycol or urea), and a quaternary ammonium salt acting
as a hydrogen bond acceptor (HBA, e.g. choline chloride, ChCl).
The intermolecular interactions between these components
provide specific properties for these neoteric solvents. Among
their advantages, it can be highlighted that they can easily be
prepared from cheap precursors, and they are composed of
natural, and therefore degradable, constituents. Their
biocompatibility with redox enzymes has been well
documented.[11] DESs have been mostly applied with
[
[
a]
b]
F. Z. Ibn Majdoub Hassani, Prof. S. Amzazi, Prof. J. Kreit
Biochemistry and Immunology Laboratory, Faculty of Sciences
Mohammed V University
BP 1014, Avenue Ibn Batouta Agdal, Rabat 10090, Morocco
F. Z. Ibn Majdoub Hassani, Prof. I. Lavandera
Organic and Inorganic Chemistry Department
University of Oviedo
Avenida Julián Clavería 8, 33006 Oviedo, Spain
E-mail: lavanderaivan@uniovi.es
(
multienzymatic) whole cell systems to modify the
stereoselectivity of bioreduction processes, due to the specific
inhibition of some of the enzymes present in the cell.[ Also, they
Supporting information for this article is given via a link at the end of
the document.
12]
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ChemCatChem
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have been found as suitable media for preparations which
overexpress ADHs,[13] and even for nicotinamide cofactor
recycling purposes.[14]
Table 1. Bioreduction of halogenated ketones 1-8a (20 mM) in the presence
of DES (50% v/v).
[a]
To the best of our knowledge, there is just one report that
describes the bioreduction of an α-halogenated ketone using
whole cells in a medium containing DESs.[15] Herein, we have
focused on the development of efficient reduction processes by
utilizing overexpressed ADHs to synthesize various enantiopure
halohydrins, key precursors of biologically active compounds,
studying the influence in the reaction mixture of a eutectic solvent
composed of choline chloride and glycerol (1:2 mol/mol).
Results and Discussion
Entry
Ketone
ADH
Conv (%)[b]
ee (%)[b]
In the present study, we used lyophilized preparations of E. coli
overexpressing the (R)-selective ADH from Lactobacillus brevis
1
2
3
4
5
6
7
8
9
1a
1a
2a
2a
3a
3a
4a
4a
5a
5a
6a
6a
7a
7a
8a
8a
ADH-A
LBADH
ADH-A
LBADH
ADH-A
LBADH
ADH-A
LBADH
ADH-A
LBADH
ADH-A
LBADH
ADH-A
LBADH
ADH-A
LBADH
>99
>99
>99
>99
>99
>99
>99
88
>99 (R)
>99 (S)
>99 (R)
>99 (S)
>99 (R)
>99 (S)
>99 (R)
>99 (S)
>99 (R)
>99 (S)
>99 (R)
>99 (S)
>99 (R)
97 (S)
16]
(
(
LBADH)[ and the (S)-selective ADHs from Rhodococcus ruber
ADH-A),[17] Thermoanaerobacter ethanolicus (TeSADH),[18] and
Thermoanaerobacter sp. (ADH-T).[19] These preparations were
employed due to their advantages regarding other enzymatic
formulations in terms of higher stability and easy handling.[20]
From a previous work in our research group,[13a] we already knew
that the ChCl:glycerol (1:2 mol/mol) DES mixture was compatible
with these ADHs. Also, this mixture presents an adequate
viscosity, therefore no mass-transfer limitations were expected
even at large DES concentrations in the reaction medium.
Therefore, we focused on the bioreduction of 8 halogenated
96
10
11
12
95
ketones (1-8a, Table 1), in the presence of this eutectic mixture at
21]
2
0% and 50% v/v in Tris-SO
4
buffer[ 50 mM pH 7.5 (Table 1 and
>99
>99
>99
97
Tables S2-S9 in the Supporting Information). In order to recycle
the nicotinamide cofactor (1 mM), propan-2-ol was used as
hydrogen donor in a “coupled-substrate” approach.[22] The
reduction of α-halogenated carbonyl compounds is favored
thermodynamically.[23] Therefore, it is not necessary to employ an
elevated molar excess of the co-substrate to achieve quantitative
conversions.
13
14
15
>99
>99
>99 (R)
>99 (S)
16
From the initial enzyme screening with substrate 1a (Table S2
in the Supporting Information), it was clear that the best choices
[
a] For reaction conditions, see the Experimental Section. [b] Measured by
to
accomplish
the
reduction
processes
were
chiral GC. There is a change in the CIP priority.
stereocomplementary ADH-A and LBADH.[16] It must be noted
that ADH-A afforded the (R)-alcohols while LBADH the (S)-
antipodes. This is simply due to a change in the Cahn-Ingold-
Prelog (CIP) priority of the alcohol substituents. The absolute
configuration of these alcohols was assigned based on previous
examples described in the literature and also by comparison of
the GC retention times and optical rotation values (for more
information, see Section 4 in the Supporting Information). As can
be seen in Table 1, both ADHs catalyzed the bioreductions in the
presence of a high amount of DES (50% v/v) in the reaction
medium, providing the desired aromatic or aliphatic halohydrins
[17]
Due to the fact that the substrate concentration in these reduction
protocols was still modest (20 mM), we decided to increase it
while maintaining the same amount of the biocatalyst and the
cofactor in the experiments. We usually performed the
bioreductions at 20% v/v and 50% v/v of DES, and compared the
results with the reactions in plain buffer. As shown in Tables S2-
S9 in the Supporting Information, in most cases the ketone
concentration could be effectively increased up to 300-400 mM
when employing 20% v/v of DES. However, slightly lower
conversions were observed at these substrate concentrations in
the presence of 50% v/v of DES. Meanwhile, the stereoselectivity
of all these reactions remained elevated for both ADHs, so that
the synthesis of both halohydrin enantiomers was feasible. For
comparison, the same transformations were performed in plain
buffer, obtaining in some cases comparable results, but for some
reductions better conversions were achieved in the presence of
(
R)- or (S)-1-8b with excellent conversions and enantiomeric
excess (ee). Due to the mildness of these processes, no by-
products (e.g., epoxides) were detected in the reaction mixtures.
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DES, as shown in Figure 2. We think that the cosolvent can be
beneficial in terms of a better substrate solubility and therefore
mass transfer. An advantage of employing lyophilized whole cells
is that the cell walls are partially disrupted and therefore the
substrate can be more available for the in-cell enzyme.
Table 2. Bioreduction of halogenated ketones 1-8a (200 mM) with E.
coli/ADH-A in the presence of DES (20% v/v) and without external addition
of the cofactor NAD .
+
[a]
Entry
Ketone
Conv (%)[b]
ee (%)[b]
1
2
3
4
5
6
7
8
1a
2a
3a
4a
5a
6a
7a
8a
82
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
71
>99
97
>99
97
>99
97
[
a] For reaction conditions, see the Supporting Information. [b] Measured by
chiral GC.
In order to demonstrate the applicability of this methodology,
ADH-catalyzed transformations in the presence of DES were
performed at a preparative scale (Figure 3). This way, 250 mg of
target ketones 1-8a and an extra experiment with substrate 4a at
1
g-scale were performed to synthesize the corresponding
enantiopure halohydrins. The products could easily be isolated
after a liquid-liquid extraction protocol in high yields.
Conclusions
This contribution demonstrates that eutectic mixtures can improve
the productivity in oxidoreductase-mediated transformations to
synthesize relevant chiral halohydrins at high concentrations.
Lyophilized E. coli whole cells overexpressing ADHs have been
used in a mixture of aqueous buffer and DES (ChCl: glycerol 1:2
mol/mol) up to 50% v/v. This medium did not affect negatively the
activity and selectivity of two stereocomplementary ADHs, namely
Lactobacillus brevis ADH and Rhodococcus ruber ADH, obtaining
better results in the presence of 20% v/v of the cosolvent. Hence,
the bioreductions of various α-halogenated ketones could be
performed up to 300-400 mM achieving high conversions (usually
Figure 2. Comparison between conversions for some selected bioreductions
with: A) E. coli/ADH-A; and B) E. coli/LBADH in plain buffer (blue bars) and in
the presence of ChCl:glycerol (1:2 mol/mol) DES at 20% v/v (green bars). In all
cases, the obtained ee values were >99%.
The cofactor is already present in the whole cell preparation, and
although in lyophilized E. coli cells it can diffuse to the reaction
medium, its external addition could not be of absolute necessity,
as the biotransformation occurring favors the diffusion inside the
cells. In a set of experiments, the bioreductions in the presence
of DES were performed at 200 mM ketone concentration without
adding the nicotinamide cofactor. While for NADP-dependent
LBADH, the results were not satisfactory (data not shown), for
NAD-dependent ADH-A, high conversions were achieved,
especially when using 20% v/v of DES (Table 2 and Table S10 in
the Supporting Information). This can be explained by the fact that
>90%) and excellent enantiomeric excess (>99%). The possibility
of avoiding the external addition of the cofactor NAD in ADH-A-
mediated reductions was also feasible, performing the reactions
at 200 mM. These biotransformations were scaled up to 1 g giving
access to a series of enantiopure halohydrins. Overall, the
present study shows a good example where the combination of
DESs with lyophilized whole cell systems overexpressing a
biocatalyst can be beneficial in a biotransformation and shows the
great potential that these cheap and environmentally friendly
solvents can possess for biotechnological applications.
+
there is a higher cytosolic concentration of NADH/NAD with
respect to NADPH/NADP+ in E. coli cells.[24]
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Figure 3. Preparative transformations at 250 mg (black color) and 1 g-scale (blue color) to synthesize enantiopure halohydrins in the presence of DES.
supernatant of the bioreduction mixture was collected by centrifugation
and transferred into an extraction funnel. The deposited pellet was washed
with ethyl acetate (30 mL) to extract the residual substrate and product in
the Falcon tube by centrifugation, and it was pooled into the funnel with
Experimental Section
the previous organic phase. This organic solvent was washed with water
General procedure for the enzymatic reduction of halogenated
ketones in the presence of a deep eutectic solvent
(20 mL). The phases in the funnel were separately collected. The aqueous
phase was again extracted with ethyl acetate (50 mL). This organic phase
was combined with the previous one to yield a combined extract that was
washed with water (10 mL, to eliminate the residual glycerol), collected in
In a 1.5 mL-Eppendorf vial, the reaction mixture (final volume: 0.6 mL),
contains: 10 mg of the corresponding E. coli/ADH lyophilized cells, Tris-
an Erlenmeyer flask, dried over Na
2 4
SO and filtered into a round-bottom
SO
4
buffer 50 mM pH 7.5, NAD(P)+ (1 mM), DES (ChCl: glycerol 1:2
flask. Na SO was washed with ethyl acetate (10 mL). The solvent was
2
4
mol/mol, 20% or 50% v/v), propan-2-ol (5-15% v/v) and the halogenated
ketone (1-8a, Table 1, 20-500 mM), dissolved in DMSO. The final amount
of DMSO in the assay was between 2.5-5% v/v. The reduction reactions
were carried out during 24 h, at 30 ºC in an orbital shaker at 250 rpm. The
crude was extracted with ethyl acetate (2 x 500 μL). The organic layers
evaporated under reduced pressure, affording the alcohol (R)-4b (900 mg,
89% yield) in >99% ee.
Acknowledgements
2 4
were collected by centrifugation, combined, and dried over Na SO .
Conversion rates and enantiomer excess of the halohydrins were
determined by GC on a chiral column (see the Supporting Information). To
determine the ee values for halohydrins 7b and 8b, an acetylation step
Financial support from the Spanish Ministry of Economy and
Competitiveness (MEC, Project CTQ2016-75752-R) is gratefully
acknowledged. We thank Prof. Wolfgang Kroutil (University of
Graz, Austria) for providing us with alcohol dehydrogenases
overexpressed in E. coli cells.
was required. 4-Dimethylaminopyridine (DMAP,
1 mg) and acetic
anhydride (30 μL) were mixed and then added to a volume of 200 μL of
the extracted reaction crude solution. The mixture was shaken for 3 h at
3
0 °C, and then quenched with 0.5 mL water for 30 min. The acetylated
alcohol was extracted with ethyl acetate (2 x 500 μL), and the combined
organic phases were dried over Na SO , and subjected to GC analysis.
2
4
Keywords: deep eutectic solvents • alcohol dehydrogenases •
bioreduction • selectivity • halohydrins
Scale-up of the bioreduction of 4a (1 g, 300 mM) in the presence of
DES (20% v/v)
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ChemCatChem
10.1002/cctc.201901582
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A deep eutectic solvent composed of
choline chloride:glycerol (1:2 mol/mol)
has been used as cosolvent in the
Fatima Zohra Ibn Majdoub Hassani,
Saaid Amzazi, Joseph Kreit, and Iván
Lavandera*
alcohol
bioreduction of
halogenated ketones
lyophilized E. coli cells overexpressing
the enzyme. Various halohydrins were
obtained at high concentration (up to
dehydrogenase-catalyzed
series of α-
employing
a
Page No. – Page No.
Deep Eutectic Solvents as Media in
Alcohol Dehydrogenase-Catalyzed
Reductions of Halogenated Ketones
500 mM) with high conversions and
enantiomeric excess
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