Programming cascade reactions interfacing biocatalysis with transition-metal catalysis in: Deep Eutectic Solvents as biorenewable reaction media

Article abstract of DOI:10.1039/c8gc00861b

The first application of Deep Eutectic Solvents (DESs) in the asymmetric bioreduction of ketones has been accomplished for purified ketoreductases (KREDs). The performance of the biocatalysts was enhanced by increasing the percentage of neoteric solvent in DES-buffer mixtures. At a buffer content of 50% (w/w) and even 20% (w/w), the combination of either choline chloride (ChCl)/glycerol (Gly) (1:2) or ChCl/sorbitol (1:1) proved to be most effective for achieving up to >99% conversion and up to >99% enantiomeric excess of the corresponding secondary alcohols. Moreover, this reaction medium was used to perform the first example of a chemoenzymatic cascade process in DES-buffer mixtures, namely the ruthenium-catalysed isomerisation of racemic allylic alcohols coupled with a further enantioselective bioreduction, in both sequential and concurrent modes.

Full text of DOI:10.1039/c8gc00861b

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Programming Cascade Reactions Interfacing Biocatalysis with
Transition-Metal Catalysis in Deep Eutectic Solvents as Biorenewable
Reaction Media†
a
b
c
b
a
Luciana Cicco, Nicolás RíosꢀLombardía, María J. RodríguezꢀÁlvarez, Francisco Morís, Filippo M. Perna,
a,
c,
b,
Vito Capriati, * Joaquín GarcíaꢀÁlvarez * and Javier GonzálezꢀSabín *
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
The first application of Deep Eutectic Solvents (DESs) in
asymmetric bioreduction of ketones has been accomplished
for purified ketoreductases (KREDs). The performance of the
biocatalysts was enhanced by increasing the percentage of
neoteric solvent in DES-buffer mixtures. At buffer content of
Along with these properties, DESs have unexpectedly opened
the floodgates to new perspectives and broad applications in i)
polar organometallic chemistry (organolithium and Grignard
reagents), thereby contributing to build new bridges between
9
10
main group chemistry and green chemistry, and ii) metalꢀ, bioꢀ
11
12
,
and organocatalysed transformations. Tremendous and
5
0% (w/w) and even 20% (w/w), either the combination of
choline chloride (ChCl)/glycerol (Gly) (1:2) or ChCl/sorbitol
1:1) proved to be most effective for achieving up to >99%
revolutionary advances have also been recently made in the field
of biocatalysis with several ad hoc protocols set up for
biotransformations catalysed by both isolated enzymes (lipases,
proteases, epoxide hydrolases, peroxidases and lyases) and whole
(
conversion and up to >99% enantiomeric excess of the
corresponding secondary alcohols. Moreover, this reaction
medium was used to perform the first example of
chemoenzymatic cascade process in DES-buffer mixtures,
namely the ruthenium-catalysed isomerisation of racemic
allylic alcohols coupled with a further enantioselective
bioreduction, in both sequential and concurrent mode.
11
cells in DESs and DESꢀbuffer mixtures. To the best of our
knowledge, however, the use of DESs as effective reaction media
in bioreduction processes has been limited to date to whole
11c,e,h,kꢀp
cells.
A fascinating and intriguing switch in the rate of
reaction and enantioselectivity was recently found in baker’s
11c
yeastꢀmediated reduction of both βꢀketoesters
containing ketones
and arylꢀ
11h
by simply changing the solvent from water
Introduction
to DESꢀwater mixtures. Several whole cells overexpressing
oxidoreductases were also screened towards arylꢀcontaining
ketones in DESꢀwater mixtures for the synthesis of chiral key
building blocks and Active Pharmaceutical Ingredients
As illustrated by the exponential growth of literature over the past
decade, considerable attention has been focused on Deep Eutectic
Solvents (DESs) as an increasingly valuable alternative to volatile
organic compounds from the standpoint of “greenness” in several
11h,k
(APIs).
The design of tandem metalꢀ and bioꢀcatalysed
organic processes is also a burgeoning field mainly investigated
in water, the natural medium of enzymes. Apart from some
1
fields of sciences such as electrochemistry and metal processing,
2
3
4
6
organic synthesis, material chemistry, nanotechnology,
13
exceptions, however, these catalytic networks typically suffer
5
photosynthesis and energy technology, separation processes,
from several drawbacks such as the compatibility of the involved
catalysts with reaction conditions or undesired crossꢀreactivity. In
addition, the solubility of most organic substrates is usually low
in water, which limits the maximum substrate concentration with
7
and stabilisation of DNA. These eutectic mixtures are usually
made from a salt with a cation capable of forming hydrogen
bonding and a neutral hydrogen bonding donor molecule, thereby
forming an extensive Hꢀbond network throughout the solvent
which stabilises liquid configurations and hence results in a large
melting point depression with respect to the melting temperature
of the individual components. DESs share many physicochemical
properties with conventional ionic liquids (ILs) (e.g., thermal
stability, low vapour pressure, nonꢀflammability, easy of
14
the prospect of meeting industrial criteria.
Building on our interest in bioreductions and in DES for
11h,k,15
exploring novel paradigms in biocatalysis,
present the first successful reaction of purified ketoreductases
KREDs) in the asymmetric bioreduction of arylꢀcontaining
prochiral ketones directly in DESꢀbuffer mixtures as sustainable
herein we
(
1
recycling), but are cheaper, easier to make (just by heating or
16
reaction media. Furthermore, in this work we also describe the
first example of oneꢀpot chemoenzymatic cascade by interfacing
a metalꢀcatalysed isomerisation reaction of allylic alcohols with a
enantioselective KREDꢀpromoted bioreduction in aqueous buffer
eutectic mixtures both in sequential and in concurrent fashion.
gently warming), do not require further purification, offer high
tunability, and are believed to be more biodegradable and less
toxic compared to traditional ILs due to the use of
8
environmentally friendly substances.
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DOI: 10.1039/C8GC00861B
+
Thus, a practical approach to convert a racemic mixture of allylic
alcohols into a stereodefined, saturated enantiopure secondary
alcohol (R or S) has been set up without isolation/purification
steps. The overall transformation involves three consecutive
steps: i) reduction of the allylic C–C double bond, ii) oxidation of
the secondary alkyl/aryl carbinol moiety, and iii) enantioselective
bioreduction of the inꢀsitu generated prochiral ketone (Scheme 1).
NADP ) from 20 to 50% (w/w) (Table 1). In a typical experiment
aimed at evaluating the enzymatic performance, 1a (20 mM) was
incubated in a mixture of DES and KPi buffer (15% w/w of iꢀ
PrOH) at 30 ºC and 250 rpm during 24 h. As reported in Table 1,
most of the employed KREDs led to poor conversions in
1ChCl/2H O at 50% and 80% (w/w) DES, and proved to be even
2
inactive in ChClꢀbased DESs containing urea or lactic acid as the
hydrogen bond donor (HBD) at 50% (w/w) DES. Gratefully,
DESs containing sorbitol or glycerol as HBD and ChCl as the
hydrogen bond acceptor resulted in high conversions, in
particular at 50% (w/w) DES. Moving to 80% (w/w) DES, more
than half of KREDs still remained very active in 1ChCl/1Sorb (c
>80%; Table 1, entries 2–7,10), but only KREDꢀP2ꢀC11
displayed excellent activity both in 1ChCl/1Sorb and
R2
OH
R4
R2
R2
OH
∗
O
[
Ru]cat
KRED
R1
R1
R4
R1
R4
DES-buffer
DES-buffer
i-PrOH
R3
R3
R3
(R) or (S)
One-pot metal- and bio-catalysis
Scheme 1. Oneꢀpot Ruꢀcatalysed isomerisation of allylic alcohols
combined with an enantioselective bioreduction in DESꢀbuffer medium.
1
ChCl/2Gly, thereby enabling complete conversion (c >99%) at
Results and Discussion
50% and 80% (w/w) DES (Table 1, entry 5). Recently, the
commercial supplier of KREDs unveiled the source of some
enzymes contained in the kit: 16 out of 24 KREDs variants (six of
KRED-catalysed reduction of ketones in DES-buffer mixtures
1
8
In order to explore the viability of eutectic mixtures in the
bioreduction of prochiral aromatic ketones, we set out to
investigate as a bench reaction the bioreduction of propiophenone
which are included in Table 1; see also details in the ESI) have
been derived from the shortꢀchain dehydrogenase of the
19
bacterium Lactobacillus kefiri (LKADH). For the sake of
comparison, this overexpressed enzyme was similarly submitted
to the same panel of experiments. As can be seen in Table 1
(entry 11), L. kefiri displayed good activity towards 1a at 50%
and 80% (w/w) DES (c from 80 to >99%) in the three DESs
considered, but it was inactive in those containing urea and lactic
acid.
(
1a) to 1ꢀphenylꢀ1ꢀpropanol (2a) in various DES systems
®
catalysed by a set of ten KREDs selected from the Codex KRED
Screening Kit, which are known to exhibit high activity and
enantioselectivity towards 1a.
1
5b,17
Five choline chloride (ChCl)ꢀ
based eutectic mixtures, namely 1ChCl/2Gly (Gly = glycerol),
1
1
ChCl/2H O, 1ChCl/1Sorb (Sorb = sorbitol), 1ChCl/2Urea and
ChCl/2Lac (Lac = lactic acid) were screened at buffer content
2
(
phosphate buffer 125 mM pH 7.0, 1.25 mM MgSO , 1 mM
4
a,b
Table 1 Effect of different DESsꢀbuffer media on the conversion of the KREDꢀcatalysed bioreduction of propiophenone (1a).
1
ChCl/2Gly
1ChCl/2H O
1ChCl/1Sorb
2
c
c
c
c
c
c
c
c
c
5
0%
80%
100%
DES
50%
80%
100%
DES
50%
80%
100%
DES
Entry
KRED
DES
DES
DES
79
53
14
ꢀ
DES
DES
DES
1
2
3
4
5
6
7
8
9
NADHꢀ110
P1ꢀA04
P1ꢀC01
P1ꢀH10
P2-C11
P2ꢀD12
P2ꢀH07
P3ꢀB03
P3ꢀG09
P3ꢀH12
LKADH
39
ꢀ
33
7
ꢀ
ꢀ
50
50
25
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
-
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
42
25
93
95
92
>99
88
82
33
35
81
ꢀ
ꢀ
ꢀ
ꢀ
-
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
>99
94
>99
>99
>99
>99
>99
>99
50
ꢀ
>99
>99
90
ꢀ
ꢀ
>99
32
ꢀ
50
ꢀ
>99
19
33
ꢀ
>99
5
>99
45
ꢀ
12
ꢀ
ꢀ
ꢀ
42
ꢀ
ꢀ
ꢀ
ꢀ
45
1
0
90
ꢀ
ꢀ
50
ꢀ
94
d
1
1
>99
93
ꢀ
>99
96
ꢀ
87
81
ꢀ
(
L. kefiri)
a
+
Reaction conditions: 1a (20 mM) in a DESꢀKPi buffer mixture (900 ꢀL, 1.25 mM MgSO
4
, 1 mM NADP ), KRED (2 mg), iꢀ
b
PrOH (190 ꢀL), 24 h at 250 rpm and 30 ºC. Conversion measured by HPLC. No conversion detected both in 1ChCl/2urea
and 1ChCl/2Lac at 50% (w/w) DES. DESꢀbuffer percentages are expressed in w/w. Reaction conditions for L. kefiri: 1a (20
mM) in a DESꢀKPi buffer mixture (900 ꢀL, 1 mM MgCl , 1 mM NADP ), L. kefir (50 U), iꢀPrOH (190 ꢀL), 24 h at 250 rpm
2
and at 30 ºC.
c
d
+
2
|
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Given that both 1ChCl/2Gly and 1ChCl/1Sorb served as
excellent eutectic mixtures for the bioreduction of 1a, we sought
to explore the scope of suitable prochiral ketone substrates (1b–f)
employing the glycerolꢀbased DES because of the because of
the high viscosity of the sorbitolꢀbased eutectic mixture
even at 40 °C (vide infra). Table 2 shows selected examples of
screening with KREDs. In most of cases, KREDs led to very
similar conversions at both 50% (w/w) DES and buffer.
Conversely, KREDꢀP2ꢀC11 was the only active enzyme at 80%
to up to 80% (w/w) (Table 2, entries 2 and 9). In addition, a
ketone such as 1e whose bioreduction had been challenging in
2
0
aqueous medium (ee up to 91%, see Table S5 in the ESI), could
now be obtained in enantiopure form (entry 9). Likewise, KREDꢀ
P2ꢀH07 (Table 2, entry 10) also exhibited a significant
performance in the bioreduction of 1e by changing the solvent
from neat aqueous buffer (2e: 32% ee) to 50% (w/w) DES (2e:
98% ee). Thus, the increase of DES percentage in the buffer
medium was generally beneficial on the enantioselectivity for all
KREDs. Parallel experiments performed in 1ChCl/1Sorbꢀbuffer
followed the same trend (see Table S7 in the ESI). A DES to
buffer ratio of 50:50 (w/w) turned out to be the optimal reaction
medium for most KREDs as it provided high conversion and high
enantioselectivity. Exceptionally, KREDꢀP2ꢀC11 was able to
work even at 80% (w/w) DES increasing even more the final ees
of the produced alcohols.
2
0
(
w/w) DES concentration, thereby allowing excellent conversion
of all tested ketones with the exception of 1f (Table 2, entries 2,
, 5, 7, 9 and 12). As for stereoselectivity, KREDs followed the
3
general trend already observed with whole cells, that is a gradual
improvement of enantioselectivity by increasing the percentage
11c,h
of DES.
KREDꢀP2ꢀC11, in particular, revealed to be an
outstanding catalyst as it enabled an enhancement of the
enantiomeric excess (ee) from 78% for 2a or 54% for 2e in
aqueous buffer to >99% by increasing the concentration of DES
a
Table 2 Selected KREDꢀcatalysed reduction of ketones 1a–2a in 1ChCl/2Glyꢀbuffer.
Neat aqueous buffer
50% w/w DES
80% w/w DES
b
c
b
c
b
c
Entry
Substrate
Product
KRED
c (%)
99
>99
99
>99
99
>99
99
ee (%)
c (%)
ee (%)
c (%)
ee (%)
O
1
2
3
4
5
6
7
8
9
P1ꢀA04
P2ꢀC11
>
>99 (R)
78 (R)
82 (R)
90 (R)
92 (R)
>99 (R)
93 (R)
99 (S)
54 (R)
32 (R)
>99
>99
98
>99 (R)
96 (R)
95 (R)
>99 (R)
96 (R)
>99 (R)
95 (R)
>99 (S)
93 (R)
98 (R)
33
>99
98
ꢀ
>99 (R)
>99 (R)
97 (R)
1a
P2ꢀC11
P2ꢀH07
>
96
ꢀ
98 (R)
ꢀ
O
P2ꢀC11
P2ꢀH07
>
98
98
ꢀ
95
1c
MeO
P2ꢀC11
P3ꢀH12
>
>99
99
>99
ꢀ
98 (R)
ꢀ
>99
>99
>99
OH
P2ꢀC11
P2ꢀH07
>99
>99
97
ꢀ
>99 (R)
1
1
1
0
1
2
2e
ꢀ
P3ꢀH12
P2ꢀC11
>99
39
89 (S)
10 (R)
>99
6
90 (S)
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
a
+
Reaction conditions: ketone 1 (20 mM) in 1ChCl/2GlyꢀKPi buffer mixture (900 ꢀL, 1.25 mM MgSO
4
, 1 mM NADP ), KRED (2 mg), iꢀPrOH (190 ꢀL), 24 h at 250
rpm and at 30 ºC. Conversion measured by HPLC. Enantiomeric excess (ee) measured by chiralꢀphase HPLC. R or S refers to the absolute configuration of alcohols
a–2f.
b
c
2
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Stability of KREDs in DES-buffer mixtures
complete conversion despite a previous incubation of 3 h. In
summary, the studied KRED exhibited slightly lower stability in
DESs than in neat aqueous buffer, although it tolerated
temperatures up to 40 ºC.
Cell integrity is known to be preserved in DESs in the case of
whole cells, but there are no data for activity using isolated
11c,h
enzymes.
However, as transpires from the data reported in
Tables 1 and 2, all the selected, purified KREDs surprisingly
remain very active in ChClꢀbased buffer media (e.g., 1ChCl/2Gly,
1
ChCl/1Sorb). We sought to capitalize on that by getting more
insight about the stability of KREDꢀP2ꢀC11, which is the
biocatalyst exhibiting the highest activity in DESꢀbuffer media.
First, the impact of temperature was evaluated by performing the
reduction of 1a in 1ChCl/2Glyꢀbuffer 80:20 (w/w) at 30 ºC, 40
ºC, and 50 ºC (Figure 1). Bioreduction took 5 h at 30 ºC to reach
complete conversion, while the conversion was no higher than
9
0% after 1 h. An increase of the temperature to 40 ºC resulted in
an acceleration of the process, taking now only 2 h for 1a
consumption (c = 98% after 1 h). On the other hand, a
temperature as high as 50 ºC had a detrimental effect in spite of a
c = 43% after 1 h; the conversion stayed at 50% after 24 h not
evolving further. A similar outcome was observed for the
reduction of 1a catalysed by KREDꢀP2ꢀC11 in 1ChCl/1Sorbꢀ
buffer 80:20 (w/w) with quantitative biotransformations at 30 ºC
or 40 ºC but, again, poor conversion after 24 h at 50 ºC (c =
Figure 2. Kinetics of reduction of 1a catalysed by KREDꢀP2ꢀC11 at 40
ºC with KRED incubated in the medium [(1ChCl/2Glyꢀbuffer 80:20
(w/w)] prior to the addition of 1a; Blue: 1 h; Red: 2 h; Green: 3 h.
One-pot ruthenium-catalysed allylic alcohol isomerisation-
asymmetric bioreduction in DES-buffer mixtures
3
0%).
In the last years, the rutheniumꢀcatalysed isomerisation of
allylic alcohols has been efficiently accomplished not only in
21
water but also in other unconventional solvents such as ionic
22
23
10f
liquids, glycerol, and DESs. What is more important, this
metalꢀcatalysed reaction could be successfully coupled with other
13b,15b,24
biotransformations
as well as with organometallic
reactions (which are typically restricted to anhydrous solvents) in
25
a oneꢀpot process. We questioned whether the excellent activity
displayed by KREDs in the reduction of ketones in neoteric
solvents (vide supra) could be exploited for setting up a oneꢀpot
isomerisation of allylic alcohols coupled with an asymmetric
KREDꢀmediated enzymatic reduction in a DESꢀbuffer mixture.
Figure 1. Kinetics of reduction of 1a in 1ChCl/2Glyꢀbuffer 80:20 (w/w)
We began our studies focusing on a sequential oneꢀpot twoꢀsteps
catalysed by KREDꢀP2ꢀC11; Blue: 30 ºC; Red: 40 ºC; Green: 50 ºC
15b,24
methodology as previously reported.
The metalꢀcatalysed
isomerisation of αꢀvinylbenzyl alcohol 3a was investigated under
the optimised conditions found for the bioreduction of ketones in
DESꢀbuffer mixtures (see Table 2) working at 200 mM substrate
concentration in 1ChCl/2Glyꢀbuffer 50:50 (w/w), at 50 ºC and
Alternatively, the reduction of 1a was also carried out in
ChCl/2Glyꢀbuffer 80:20 (w/w) at 40 ºC, but incubating for 1 h, 2
1
h and 3 h the KRED in the reaction medium prior to the addition
of the ketone. As depicted in Figure 2, the catalyst was readily
deactivated in 2–3 h, which led to poor conversion (up to 25%)
after 24 h. However, the residual activity in the KRED (despite
concomitant inactivation) after 1 h of incubation was enough to
reach a conversion of 93% after only 1 h of reaction, without
further evolution. KREDꢀP2ꢀC11 remained very active in a
ChCl/1Glyꢀbuffer 80:20 (w/w) even after incubation at 40 ºC for
4 h after the addition of ketone 1a leading eventually to a
conversion of 93%. For the sake of comparison, the same set of
bioreduction of 1a by KREDꢀP2ꢀC11 was run in neat aqueous
buffer: the biocatalyst turned out to be stable at 50 ºC and led to
26,27
using 5 mol% of ruthenium complex 4 as catalyst
(Scheme 2).
Once the isomerisation was complete, KREDꢀP1ꢀA04 and
+
NADP were sequentially added without isolating the
intermediate ketone 1a (Scheme 2), and the resulting mixture
stirred for 24 h at 30 ºC (Table 3, entry 1). Saturated alcohol (R)ꢀ
2
a was obtained with quantitative conversion of substrate,
1
2
excellent isolated yield (95%), and ee >99%. Following an
identical protocol, racemic allylic alcohols 3b–d gave rise to the
corresponding (R) or (S)ꢀsaturated alcohols 2b–d with very high
ees (Table 3, entries 3, 5 and 7) by selecting an adequate KRED.
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The overall methodology is operationally simple and excellent
levels of both conversion (>90%) and enantioselection (>99%) in
the final products have been achieved. On the other hand, the
same sequential processes starting from 3a–d and accomplished
with L. kefiri provided lower conversion values and
enantioselectivies than those measured with purified KREDs
aqueous buffer, that is with the two catalytic systems coexisting
from the outset, an open issue was the stability of the KRED in
15b
the reaction medium. These enzymes, indeed, suffered from
rapid deactivation, thereby leading to moderate overall
conversions in the case of allylic alcohols undergoing slow
isomerisation. We selected the 1ChCl:2Glyꢀbuffer 80:20 (w/w)
mixture as the reaction medium for the transformation of the
vinylic alcohol 3a into 2a, which was catalysed simultaneously
and competitively by both KREDꢀP2ꢀC11 and complex 4. The
load of 4 was optimised to 10 mol%. Indeed, under these
conditions, the isomerisation of 3a–d is completed in less of 2 h,
and is fast enough to preserve the enzyme’s lifetime (see Table
S8, ESI). Thus, allylic alcohol 3a was incubated at 40 ºC and 250
rpm in the above DESꢀbuffer medium containing both catalysts.
After 24 h, HPLCꢀanalysis revealed that the starting racemic
allylic alcohol 3a was completely consumed and the target
saturated alcohol (R)ꢀ2a obtained with an overall conversion of
90% and an ee >99%. The extension of the above protocol to
substrates 3b–d furnished the saturated chiral alcohols 2b–d with
overall conversion ranging from 68 to 96% for the Rꢀconfigured
(Table 3, entries 2, 4, 6 and 8). The only exception was 3a, which
led quantitatively to its saturated analogue (R)ꢀ2a in enantiopure
form (Table 3, entry 2). These results are consistent with a
compatibility of KREDs with the reaction medium coming from
the metalꢀcatalysed step, the impact of the Ru(IV) catalyst 4 on
the enzymatic performance being negligible. Although
15b
comparable results have been obtained in pure water,
the
advantage of using DESꢀbuffer mixtures arises from the
beneficial effects exerted by such neoteric solvents on the
enantioselectivity of KREDs, particularly in the case of
challenging substrates unresponsive to be stereoselectively
reduced in neat aqueous buffer.
2
8
enantiomer with an ee >99% in all cases (Table 4, entries 1ꢀ4).
Conversely, after running concurrent process with the
a
overexpressed LKADH of L. kefiri and 3a, we noticed that
unsaturated alcohol 3a remained partially unreacted, as though
enzyme exerted some kind of inhibition on the metal complex 4
(Table 4, entry 5). HPLC analysis of the processes carried out
with purified KREDs revealed that 4 isomerised quantitatively
the starting allylic alcohol in all cases. Thus, the final ketone to
saturated alcohol ratio found most probably is related to the
significant difference in the catalytic performance exhibited by
isolated KREDs according to the nature of the in situ formed
ketone. Remarkably, the biotransformation of 3d provided
enantiopure (R)ꢀ2d in an overall conversion of 96% (Table 4,
entry 4), which improved previous results in aqueous buffer
Scheme 2. Oneꢀpot cascade synthesis of optically active alcohols by Ruꢀ
catalysed isomerisation/ enzymatic reduction in DESꢀbuffer medium in
both sequential (upper) and concurrent (lower) mode.
15b
mixtures.
a
a
Table 3 Chemoenzymatic oneꢀpot process in sequential mode.
Table 4 Chemoenzymatic oneꢀpot process in concurrent mode.
c
%)
Yield
(%)
ee
(%)
b
ee
(%)
Entry
Substrate
Product
KRED
Entry
Substr.
Prod.
Product ratio (%)
c
b
c
d
(
Allylic
alcohol
Saturated
alcohol
1
2
3
4
5
6
7
8
3a
3a
3b
3b
3c
3c
3d
3d
2a
2a
2b
2b
2c
2c
2d
2d
P1ꢀA04
L. kefiri
P2ꢀH07
L. kefiri
P3ꢀH12
L. kefiri
P2ꢀH07
L. kefiri
>99
>99
95
95
92
90
64
90
60
90
72
>99 (R)
>99 (R)
>99 (R)
93 (R)
Ketone
1
2
3
4
3a
3b
3c
2a
2b
2c
0
0
0
10
30
32
90
70
68
>99
>99
>99
70
95
>99 (S)
93 (R)
3d
3a
2d
2a
0
4
96
21
>99
>99
d
5
33
46
65
a
Reaction conditions (entries 1ꢀ4): to a solution under an Ar atmosphere of 3a–d
200 mM) in a mixture of KPi buffer 125 mM (1.25 mM MgSO ) at pH 7.0 (0.5
94
>99 (R)
98 (R)
(
4
mL), DES 1ChCl/2Gly (0.5 mL ) and iꢀPrOH (175 ꢀL), complex 4 (10 mol%),
80
+
KREDꢀP2ꢀC11 (100% w/w) and NADP (1 mM) were added, and the resulting
a
b
c
Reaction conditions: 3a–d (200 mM) was dissolved under an Ar atmosphere in a
mixture of KPi buffer 125 mM (1.25 mM MgSO ) at pH 7.0 (0.5 mL), DES
ChCl/2Gly (0.5 mL ) and iꢀPrOH (175 ꢀL). Then, complex 4 (5 mol%) was added,
and the mixture was stirred at 50 ºC. Once the isomerisation was complete, KRED
mixture was stirred for 24 h at 250 rpm and at 40 ºC; Measured by HPLC.
Enantiomeric excess (ee) measured by chiralꢀphase HPLC. The Rꢀenantiomer was
obtained in all cases. Reaction conditions (entry 5): to a solution of 3a (200 mM)
in a mixture similar to that reported for entries 1ꢀ4, complex 4 (10 mol%), L. kefiri
4
d
1
+
+
(
100% w/w) and NADP (1 mM) were added, and the mixture was shaken for 24 h at
(1000 U) and NADP (1 mM) were added, and the resulting mixture was stirred
2
50 rpm and at 30 ºC (entries 1, 3, 5 and 7). For entries 2, 4, 6 and 8, L. kefiri (1000
for 24 h at 250 rpm and at 40 ºC.
+
U) and NADP (1 mM) were added, and the mixture was shaken for 24 h at 250 rpm
and at 30 ºC. Conversion (c) measured by HPLC. The yields reported are for
products isolated and purified by column chromatography. Enantiomeric excess (ee)
b
c
d
measured by chiralꢀphase HPLC.
Conclusion
As for metalꢀcatalysed isomerisation coupled with
bioreduction processes run in a concurrent fashion in pure
To summarise, purified/overexpressed KREDs showed good
chemical stability and exhibited excellent catalytic performance
This journal is © The Royal Society of Chemistry [year]
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DOI: 10.1039/C8GC00861B
in ad hoc mixtures of Deep Eutectic Solvents and aqueous
buffers. In particular, both the 1ChCl/2Glyꢀbuffer and the
Boothby, G. Capper, D. L. Davies and R. K. Rasheed, J. Am. Chem.
Soc., 2004, 126, 9142.
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1
ChCl/1Sorbꢀbuffer mixtures turned out to be the most effective
media for carrying out several bioreductions. The higher the
percent of DES in the mixture, the greater the enantioselectivity
displayed by the selected KRED, which led to a substantial
enhancement of the ee of the resulting secondary alcohol.
Gratifyingly, the above DESꢀbuffer mixtures could be
successfully used for the first time as suitable reaction media also
for setting up a chemoenzymatic cascade process, run both in
sequential and in concurrent mode, such as the rutheniumꢀ
catalysed isomerisation of racemic allylic alcohols coupled with
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Acknowledgements
Monflier and S. Tilloy, Green Chem., 2014, 16, 3876; e) J. Lu, X.ꢀT.
Li, E.ꢀQ. Ma, L.ꢀP. Mo and Z.ꢀH. Zhang, ChemCatChem, 2014, 6,
We are indebted to the MINECO of Spain (CTQ2016ꢀ81797ꢀ
REDC and CTQ2016ꢀ75986ꢀP) and the Gobierno del Principado
de Asturias (Project GRUPIN14ꢀ006) for financial support. J.G.ꢀ
A. thanks the Fundación BBVA for the award of a “Beca
Leonardo a Investigadores y Creadores Culturales 2017”. L. C.,
F. M. P. and V. C. would like to thank the Interuniversities
Consortium C.I.N.M.P.I.S. for partially supporting this work.
Authors also thank Harald Gröger, Bielefeld University, for the
generous gift of the LHADH from Lactobacillus kefiri.
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2 3
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27 The [Ru]cat 4 catalyst ([Ru(η :η ꢀC10H16)Cl(κ ꢀ(O,O)ꢀO CCH )]) has
2 3
3
3
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6
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A tandem enzymeꢀorganocatalysed cross aldol reaction with
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2
0
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For a parametric study of the catalytic activity of the panel of KREDs
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Transition-metal complexes and enzymes play together in eutectic mixtures: An
environmentally friendly one-pot/one-step protocol for the synthesis of enantiopure
alcohols in Deep Eutectic Solvents is reported, establishing a new bridge between
transition metal-catalysed and biocatalysed organic reactions.