Biocatalysed reductions of α-ketoesters employing CyreneTM as cosolvent

Article abstract of DOI:10.1080/10242422.2021.1887150

The search for novel reaction media with environmental friendly properties is an area of great interest in enzyme catalysis. Water is the medium of biocatalysed processes, but due to its properties, sometimes the presence of organic (co)solvents is required. Cyrene^TM represents one of the newest approaches to this medium engineering. This polar solvent has been employed for the first time in biocatalysed reductions employing purified alcohol dehydrogenases. A set of α-ketoesters has been reduced to the corresponding chiral α-hydroxyesters with high conversions and optical purities, being possible to obtain good results at Cyrene contents of 30% v/v and working at substrate concentrations of 1.0 M in presence of 2.5% v/v of this solvent. At this concentration, the presence of Cyrene has a beneficial effect in the bioreduction conversion.

Full text of DOI:10.1080/10242422.2021.1887150

Biocatalysis and Biotransformation
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Biocatalysed reductions of α-ketoesters employing
TM
Cyrene as cosolvent
Gonzalo de Gonzalo
To cite this article: Gonzalo de Gonzalo (2021): Biocatalysed reductions of α-
TM
ketoesters employing Cyrene as cosolvent, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2021.1887150
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2021.1887150
RESEARCH ARTICLE
TM
Biocatalysed reductions of a-ketoesters employing Cyrene as cosolvent
Gonzalo de Gonzalo
Departamento de Qu ꢀı mica Org ꢀa nica, Universidad de Sevilla, Sevilla, Spain
ABSTRACT
ARTICLE HISTORY
The search for novel reaction media with environmental friendly properties is an area of great
interest in enzyme catalysis. Water is the medium of biocatalysed processes, but due to its prop-
erties, sometimes the presence of organic (co)solvents is required. Cyrene represents one of
Received 28 December 2020
Revised 17 January 2021
Accepted 2 February 2021
TM
the newest approaches to this medium engineering. This polar solvent has been employed for
the first time in biocatalysed reductions employing purified alcohol dehydrogenases. A set of
a-ketoesters has been reduced to the corresponding chiral a-hydroxyesters with high conver-
sions and optical purities, being possible to obtain good results at Cyrene contents of 30% v/v
and working at substrate concentrations of 1.0 M in presence of 2.5% v/v of this solvent. At this
concentration, the presence of Cyrene has a beneficial effect in the bioreduction conversion.
KEYWORDS
Biobased solvents; alcohol
dehydrogenases; biocataly-
sis; solvent engineering;
hydroxyesters; bioreductions
GRAPHICAL ABSTRACT
Commercial ADHs
Aqueous Buffer
Cyrene (up to 30% v/v)
O
O
TM
n
n
OR
OR
O
NAD(P)H
OH
X
X
1
0 mM - 1.0 M
(R)- or (S)-alcohol
High conversions
High optical purities
R: Me, Et.
X: H, CN, Cl.
n: 0, 2.
1
. Introduction
Gotor-Fern ꢀa ndez and Paul 2019). But these two
approaches do not represent a proper alternative and
a requirement of organic solvents with the same prop-
erties and a much lower environmental footprint as
the biobased solvents, that is those solvents obtained
from natural resources (Lomba 2019; Sheldon 2019),
has appeared in the recent years. The use of 2-methyl-
tetrahydrofuran (2MeTHF) (Pace 2012) or cyclopentyl
methyl ether (CPME) (de Gonzalo 2019) in biocatalyzed
reactions has been documented in several examples,
as well as other biobased solvents with interesting
applications in Green Chemistry (Jerome and Luque
In the last few years, the development of processes
catalyzed by biological systems as purified enzymes or
whole cells has experienced a great development (de
Gonzalo and Dom ꢀı nguez de Mar ꢀı a 2017; Devine 2018;
Sheldon and Woodley 2018). Water is the natural
medium in which these reactions are performed. But
this solvent can present some drawbacks for the appli-
cation of biocatalysts in organic reactions. Thus, water
is a very polar solvent, which generates solubility
issues. In addition, some side-reactions (hydrolysis) can
occur in aqueous media. By these reasons, since the
seminal discoveries by Klibanov et al. in the 80 s of
the last century, organic solvents have appeared as a
valuable reaction medium for carrying out biocata-
lyzed procedures (Zaks and Klibanov 1985; Carrea and
Riva 2000). Environmental concerns have pushed for
the developed of greener alternatives to the classical
organic solvents, including for instance the use of neo-
teric or supercritical solvents (Hern ꢀa iz 2010; Itoh 2017;
2
017; Iemhoff et al. 2018). One recent example of this
biobased approach is dihydrolevoglucosenone
Cyrene ), a polar aprotic solvent similar to N,N-dime-
TM
(
thylformamide (Sherwood 2014). Cyrene can be
obtained from natural sources, as it can be produced
by pyrolysis and/or hydrogenation of cellulose (Kudo
2017). Cyrene presents a high solubility in water, form-
ing a set of derivatives in this solvent whose
CONTACT Gonzalo de Gonzalo
gdegonzalo@us.es
Departamento de Qu ꢀı mica Org ꢀa nica, Universidad de Sevilla, Sevilla, Spain.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
G. DE GONZALO
proportions will modify the polarity of the reaction
media, being able by this reason to solve a wide set
of compounds (de Bruyn 2019). This fact can also have
effect on the catalytic processes in which Cyrene is
employed as (co)solvent. Until nowadays, very few
examples of the Cyrene application as (co)solvent in
biocatalysis have been shown. In 2020, Guajardo and
Dom ꢀı nguez de Mar ꢀı a have developed the lipase-cata-
lyzed esterification of benzoic acid and glycerol in
presence of Cyrene concentrations up to 40% v/v
14.9 min; tR (1b); 15.3 min; tR (2a): 14.3 min; tR (2b):
14.8 min; tR (3a): 18.1 min; tR (3b): 18.8 min; tR (4a):
16.8 min; tR (4b): 17.3 min; tR (5a): 14.3 min; tR
(5b): 14.5 min.
HPLC analyses were performed on a Waters 2695
Instrument equipped with a Waters 996 Photodiode
Array Detector. To determine the enantiomeric
excesses of chiral a-hydroxyesters 1–5b, a Chiralcel
OD (25 Â 0.46 cm, Daicel) column was employed, with
the following conditions: n-hexane:IPA 95:5, 1.0 mL/
ꢀ
(
Guajardo and Dom ꢀı nguez de Mar ꢀı a 2020), but no
min, 30 C. t (S)-1b: 7.8 min; t (R)-1b: 13.2 min; t (S)-
R R R
examples of its use in reductive processes catalyzed
by alcohol dehydrogenases (ADHs) have been
described. ADHs, also called ketoreductases (KREDs)
are able to catalyze the reversible reduction of car-
bonyl compounds to the corresponding chiral alcohols
in presence of nicotinamides as cofactors (Musa and
Phillips 2011; Zheng 2017; de Gonzalo and Lavandera
2
b: 8.6 min; t (R)-2b: 12.9 min; t (S)-3b: 15.8 min; t
R R R
(R)-3b: 18.1 min; t (S)-4b: 12.7 min; t (R)-4b: 15.3 min;
R R
t (S)-5b: 9.7 min; t (R)-5b: 13.8 min. The configuration
R
R
of the a-hydroxyesters were established by comparing
the HPLC chromatograms with the previously
described (Chadha and Baskar 2002).
2
020). ADHs have been widely used in synthetic meth-
2
.2. General procedure for the bioreduction of
odologies for the preparation of alcohols with differ-
ent structures. Thus, we have analysed the application
of Cyrene as cosolvent in the bioreduction of a set of
a-ketoesters to the corresponding optically active
a-hydroxyesters, which are versatile products that find
application in the chemical, food, and pharmaceutical
industries, as for instance, antibiotics and bioactive
compounds (Desage-El Murr 2003; Mallinger 2008).
ketoesters 1–5a catalyzed by ADHs using
isopropanol as cosubstrate
Unless
10–1000 mM) were dissolved in IPA (100 mL) and
Cyrene (25-300 mL). The corresponding ADH from
otherwise
stated,
a-ketoesters
1-5a
(
V
R
Codex KRED Screening Kit (10 mg) was dissolved in
KRED Recycle Mix pH 7.0 phosphate Buffer
600–875 mL) and the substrate solution was added to
P
(
ꢀ
the enzyme solution. Reactions were stirred at 30 C
and 220 rpm for 24 hours. Reactions were then
extracted with EtOAc (2 Â 0.5 mL), dried onto Na SO
2
. Experimental section
2
.1. Materials and methods
2
4
Purified alcohol dehydrogenases (CodexVR KRED
Screening Kit) were purchased from Codexis Inc.
Cyrene
available at Sigma-Aldrich. The rest of reagents and
solvents were products from Sigma-Aldrich and TCI.
Racemic a-hydroxyesters (±)-1-5 b were obtained by
reduction of the a-ketoesters employing sodium boro-
hydride in methanol with high yields (75-92%), and
exhibited physical and spectral properties in accord-
ance with those reported for (±)-1-2 b (Zhang 2014),
and the samples were directly analysed by GC/MS and
HPLC in order to determine the level of conversion as
well as the enantiomeric excesses of a-hydroxyesters
(S)- or (R)-1-5b depending on the biocata-
lyst employed.
TM
and a-ketoesters 1-5a were commercially
2.3. General procedure for the bioreduction of
ketoesters 1-5a catalyzed by ADHs using glucose/
glucose dehydrogenase as cofactor
recycling system
(±)-3-4b (Francesco 2008) and (±)-5b (Chadha and
Unless otherwise stated, ketoesters 1-5a (10–50 mM)
were dissolved in KRED Recycle Mix N Buffer pH 7.0
phosphate containing Cyrene (2.5–10% v/v) up to a
final volume of 1.0 mL. The suspension was added to
Baskar 2002).
GC/MS analyses were performed with a GC Hewlett
Packard 7890 Series II equipped with a Hewlett
Packard
5973
chromatograph
MS
(Agilent
V
R
Technologies) using a HP-5MS cross-linked methyl
the corresponding ADH from Codex KRED Screening
Kit (10 mg) and the resulting mixture was stirred at
30 C and 220 rpm for 24 hours. Reactions were then
siloxane column (30 m  0.25 mm  0.25 lm, 1.0 bar
ꢀ
N ). To monitor levels of conversion, substrates and
2
products were quantified by use of calibration curves.
The following temperature program was employed:
extracted with EtOAc (2 Â 0.5 mL), dried onto Na
SO
2 4
and the samples were directly analyzed by GC/MS and
ꢀ
ꢀ
ꢀ
5
0 C (5 min), 10 C/min to 200 C (7 min). t (1a):
HPLC in order a-hydroxyesters (R)-1-5b.
R

BIOCATALYSIS AND BIOTRANSFORMATION
3
2
2
.4. Bioreduction of ethyl benzoylformate to ethyl
-hydroxy-2-phenylacetate at multimilligram scale
bioreductions catalyzed by P2-D03 and P2-D12, being
possible to obtain the final product with complete
conversion and enantiomeric excesses around 90%
(entries 1 and 3, respectively). The use of the IPA-
dependent dehydrogenases P2-C11, P2-G03 and P1-
B02 (entries 2, 4, and 7) led to the (R)-enantiomer, but
with low or moderate enantioselectivities. Those
KREDs that required a secondary enzymatic system for
cofactor regeneration afforded the R enantiomer of
the a-hydroxyester, in most cases with optical purities
higher than 80% (entries 12, 14, and 15). The best
result was obtained with NADH 101, recovering a 98%
of (R)-1b with 88% ee. Thus, by proper selecting the
biocatalyst, it was possible to obtain both enantiomers
of 1b with high optical purities and complete conver-
sions in presence of 2.5% v/v Cyrene (Table 1).
catalysed by P2-D03 in buffer containing 2.5%
v/v cyrene
Ethyl benzoylformate 1a (1.0 M, 178.1 mg) was dis-
solved in Cyrene (25 mL) and IPA (100 mL) and added
into a suspension of P2-D03 (10 mg) in KRED Recycle
Mix P pH 7.0 phosphate buffer (875 mL). The reaction
ꢀ
was stirred for 24 hours at 30 C and 220 rpm. Water
(3 mL) was added to the reaction mixture and it was
extracted with EtOAc (6 Â 2.0 mL). The organic phases
were dried onto Na SO and solvent was removed
2
4
under reduced pressure. The crude mixture was puri-
fied by column chromatography employing n-
hexane:EtOAc 8:2 as eluent to obtain 127.9 mg of (S)-
ethyl 2-hydroxy-2-phenylacteate (1b, 71% yield) with
In view of the results obtained, P2-D03 and NADH
90% enantiomeric excess.
1
01 were further tested in the bioreduction of
a-ketoester 1a modifying some reaction parameters. It
has to be established that for these two biocatalysts
no Cyrene reduction products were observed after
3
. Results and discussion
Initial experiments were devoted to analyse the effect
of Cyrene at 2.5% v/v content on the bioreduction of a
a-ketoester as ethyl benzoylformate (1a). This process
was catalyzed by different commercially available ADHs
2
4 hours, as this solvent contains a keto group in its
structure that could be reduced by the ADHs. In a first
set of experiments the effect of Cyrene content in the
enzymatic reactions when working at 10 mM substrate
concentration was analyzed. When employing KRED
P2-D03, bioreductions can be performed using up to
V
R
(Codex KRED Screening Kit from Codexis Inc.).
Reactions were carried out using two different nicotina-
mide cofactor [NAD(P)H] recycling systems. As most of
these commercial biocatalysts were tolerant to isopro-
panol (IPA), a substrate-coupled approach for NADPH
regeneration in presence of 10% v/v of this compound
was employed. For the rest of biocatalysts tested, nico-
tinamide was recycled by an enzyme-coupled
approach, using glucose dehydrogenase (GDH) and glu-
cose as secondary enzymatic system (Scheme 1).
All the KREDs tested in presence of 2.5% v/v Cyrene
afforded optically active ethyl 2-hydroxy-2-phenylace-
tate (1 b) with complete conversion after 24 hours,
indicating that this solvent does not present any nega-
tive effect on the biocatalysts activities. Regarding the
selectivities achieved, most of IPA-dependent KREDs
led to (S)-1b with optical purities from moderate to
high. The best results were achieved in the
3
0% v/v Cyrene. At these conditions (Table 2, entry 5),
(S)-1b was still obtained with good conversion (73%)
and only a slight decrease in the product optical pur-
ity (83% ee) after 24 hours, demonstrating the good
performance of this KRED. As shown in Table 2, the
use of solvent contents from 5 to 20% v/v allowed
obtaining 1b with conversions around 90%, with no
effect in the optical purity of (S)-1b at 5–10% v/v
cosolvent (entries 2–3) and with 86% ee at 20% v/v
Table 1. Bioreductions of ethyl benzoylformate (1a) catalyzed
a
by commercial alcohol dehydrogenases from Codexis. .
b
c
Entry
ADH
Recycling
c (%)
ee (%)
Configuration
1
2
3
4
5
6
7
8
9
P2-D03
P2-C11
P2-D12
P2-G03
P2-D11
P1-C01
P1-B02
P1-B05
P1-H08
P1-B10
IPA
IPA
IPA
IPA
IPA
IPA
IPA
IPA
IPA
ꢁ97
ꢁ97
ꢁ97
ꢁ97
ꢁ97
ꢁ97
ꢁ97
ꢁ97
ꢁ97
ꢁ97
ꢁ97
ꢁ97
ꢁ97
ꢁ97
96
90
13
92
50
31
66
47
35
25
22
73
82
13
87
88
S
R
S
R
S
S
R
S
S
S
S
R
R
R
R
1
1
1
1
1
1
a
0
1
2
3
4
5
IPA
IPA
P2-C02
KRED 119
NADH 110
KRED 130
NADH 101
Glucose/GDH
Glucose/GDH
Glucose/GDH
Glucose/GDH
For reaction details, see experimental section.
b
Scheme 1. Biocatalyzed reduction of ethyl benzoylformate
Determined by GC/MS.
Determined by HPLC.
c
(1a) in buffer containing 2.5% v/v Cyrene.

4
G. DE GONZALO
Table 2. Effect of Cyrene percentage and 1a concentration in
the bioreductions catalysed by P2-D03, NADH 101 and
a
KRED 130.
b
c
Entry
ADH
[1a] (mM) Cyrene (%) c (%)
ee (%)
Config.
1
2
3
4
5
6
7
8
9
P2-D03
P2-D03
P2-D03
P2-D03
10
10
10
10
10
10
10
10
20
50
10
10
10
20
50
2.5
5
10
20
30
2.5
5
10
2.5
2.5
2.5
5
10
2.5
ꢁ97
90
90
89
86
83
88
81
60
59
41
87
87
80
86
83
S
S
S
S
S
R
R
R
R
R
R
R
R
R
R
ꢁ97
96
91
73
P2-D03
NADH 101
NADH 101
NADH 101
NADH 101
NADH 101
KRED 130
KRED 130
KRED 130
KRED 130
KRED 130
ꢁ97
68
56
53
39
ꢁ97
ꢁ97
95
88
84
1
1
1
1
1
1
a
0
1
2
3
4
5
2.5
For reaction details, see experimental section.
b
Figure 2. Bioreductions of ketoesters 2-5a catalyzed by P2-
D03 and KRED 130 in buffer containing 2.5% v/v Cyrene.
Determined by GC/MS.
Determined by HPLC.
c
bioreduction of 1a at 1.0 M with excellent results.
Thus, (S)-1b is recovered at these conditions with 90%
ee and 80% conversion (71% isolated yield) after
1
00
8
6
4
2
0
0
0
0
0
2
4 hours, with a productivity of 144.0 grams of 1 b/L
day. The conversion and productivity values were
higher than those achieved in absence of Cyrene (67%
conversion, 120.6 g/L day, 90% ee), indicating the
beneficial effect of this cosolvent at 2.5% v/v in the
enzymatic system at high substrate concentration. In
view of this result, the reaction was performed using a
higher amount of Cyrene in the reaction medium.
Thus, the bioreduction of 1.0 M of 1a in 10% Cyrene
was carried out. After 24 hours, (S)-1b was recovered
with 90% ee and 60% conversion (108.0 g/L day),
lower values than in absence of this solvent. When
working with 2.5% v/v Cyrene at lower substrate con-
centrations (250 or 500 mM), very similar conversions
and optical purities were achieved in presence and in
absence of Cyrene, with productivities between 40
and 70 g/L day, much lower than in 1.0 M of 1b.
Finally, as can be observed in Figure 1, the presence
of 2.5% v/v Cyrene in the reductions has no effect on
the optical purity of the final product at all 1a concen-
trations studied.
The same study was carried out in the bioreduc-
tions catalyzed by NADH 101, but as shown in entries
9 and 10 of Table 2, the use of 20 or 50 mM of 1a led
to a fast loss in the biocatalytic properties of this ADH.
In view of the low performance of NADH 101 when
increasing both 1a concentration or the amount of
Cyrene in the reaction medium, bireductions were also
tested in presence of KRED 130, which also led to (R)-
1b with good results (Table 2, entry 11). This biocata-
lyst seems to be more tolerant to Cyrene, as 10% v/v
of this cosolvent can be employed with a small loss in
10
20
50
100
200
1a] (mM)
250
300
400
500
1000
[
Figure 1. Effect of ethyl benzoylformate (1a) concentration in
conversion and the optical purity of (S)-1b in the bioreduc-
tions catalyzed by P2-D03 in buffer containing 2.5% v/
v Cyrene.
(
entry 4). Cyrene concentration has a more pro-
nounced effect in the bioreductions catalysed by KRED
NADH 101, as shown in entries 7 and 8. Thus, when
using only 5% v/v of the cosolvent, there is an import-
ant drop in the conversion (68% after 24 hours),
whereas the optical purity also decreased to 81% ee.
Further increase in the cosolvent concentration led to
the recovery of (R)-1b with 56% conversion and a
much lower enantiomeric excess (60% ee).
Substrate concentration was also a parameter to
analyse in the bioreduction of 1a in presence of 2.5%
v/v of Cyrene. As shown in Figure 1, P2-D03 is a very
stable biocatalyst at high substrate concentrations in
these conditions. The presence of Cyrene led to excel-
lent conversions (higher than 90%) up to 1a concen-
trations of 300 mM. The use of ethyl benzoylformate
at 500 mM concentration afforded (S)-1b with 83%
conversion, whereas it is possible to perform the

BIOCATALYSIS AND BIOTRANSFORMATION
5
the conversion, but with a more pronounced effect in
the optical purity of (R)-1b, as the a-hydroxyester was
recovered with 80% ee (entry 13). When 1a concentra-
tion was increased to 20 and 50 mM, (R)-1b was recov-
ered with conversions and optical purities around
to be a valuable cosolvent in the bioreduction of
a-ketoesters to the corresponding optically active
a-hydroxyesters catalyzed by different commercially
available alcohol dehydrogenases. When this solvent
was employed in 2.5% v/v, it is possible to obtain
both the (S)- and the (R)-hydroxyesters with complete
conversion and high optical purities depending on the
ADH employed. Bioreductions catalyzed by the IPA-tol-
erant alcohol dehydrogenase KRED P2-D03 can be car-
ried out up to 30% v/v of Cyrene with only a small
loss in the biocatalyst properties, whereas substrate
concentration can be increased up to 1.0 M, obtaining
a higher conversion in presence of this cosolvent than
in its absence and a maximum productivity of 144.0
grams of chiral alcohol produced per litre and day.
These results indicate that this biobased solvent is a
suitable option for developing further enzymatic bio-
reductions catalyzed by isolated ADHs. The search for
novel biocatalysts (both wild types or mutants) or
processes able to be conducted in sustainable solvents
must be an area of high interest for the research
groups focussed on enzyme technologies, with the
aim of developing more efficient biocatalytic proce-
dures compatible with the Green Chemistry principles.
85%, which indicates a slight loss regarding the biore-
duction at 10 mM. KRED 130 presents a higher per-
formance than NADH, but it is still sensitive to the
cosolvent amount and the substrate concentration.
Bioreductions carried out in 2.5% v/v Cyrene cata-
lysed by P2-D03 or KRED 130 were extended to other
prochiral ketoesters, as shown in Figure 2. Thus,
methyl benzoylformate (2a) was selectively reduced to
(S)-2b by P2-D03 in presence of this cosolvent with a
better result than its ethyl analogue, as after 24 hours,
a complete conversion was achieved for the (S)-
hydroxyester with 94% ee, whereas the bioreduction
catalysed by KRED 130 afforded (R)-methyl 2-hydroxy-
2-phenylacetate with 90% ee and excellent conversion.
The reactions catalysed by P2-D03 of ethyl benzoylfor-
mate derivatives presenting substituents at the aro-
matic ring, as ethyl 4-cyanobenzoylfomate (3a) or
ethyl 4-chlorobenzoylformate (4a) also afforded the
corresponding (S)-hydroxyesters with high conversions
and excellent selectivities, as shown in Figure 2. When
KRED 130 was tested as biocatalyst, both (R)-3b and
Acknowledgments
(R)-4b were recovered with conversions close to 90%
G.d.G. thanks Pablo Dom ꢀı nguez de Mar ꢀı a for his tech-
nical advice.
and high optical purities. The enzymatic processes
were also tested with an a- ketoester in which the
aromatic ring is not conjugated to the carbonyl moi-
ety, as of ethyl 4-phenyl-2-oxobutanonate (5a). This
compound led to the (S)-hydroxyester with moderate
selectivity, as (S)-5b was recovered with a moderate
optical purity (71% ee). When the reaction was per-
formed in presence of KRED 130, the (R)-enantiomer
was recovered with much better optical purity (95%
ee) and 81% conversion after 24 hours.
Disclosure statement
No potential conflict of interest was reported by
the author(s).
Funding
MINECO was acknowledged for personal funding (Ram oꢀ n y
Cajal Program).
Bioreduction of a-ketoesters 2-5a catalysed by P2-
D03 was also tested in buffer containing 2.5% v/v
Cyrene at 1.0 M substrate concentration. After
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4. Conclusion
TM
In the present paper, Cyrene , a biobased solvent
obtained from renewable sources, has demonstrated

6
G. DE GONZALO
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