A designer natural deep eutectic solvent to recycle the cofactor in alcohol dehydrogenase-catalysed processes

Article abstract of DOI:10.1039/c9gc00318e

Deep eutectic solvents (DESs) nowadays represent a sustainable alternative to traditional organic solvents in (bio)transformations. Herein, the use of a solvent composed of an aqueous buffer and choline chloride:glucose (1.5:1 mol/mol) is proposed, a natural DES (NADES) serving as both a cosolvent and efficient system to recycle the nicotinamide cofactor. Thus, glucose from the NADES served as a co-substrate required for several alcohol dehydrogenases to reduce different prochiral ketones, and also helped to solubilise the organic compounds to develop effective biotransformations at higher substrate concentrations.

Full text of DOI:10.1039/c9gc00318e

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A designer natural deep eutectic solvent to recycle the cofactor in
alcohol dehydrogenase-catalysed processes
Received 00th January 20xx,
Accepted 00th January 20xx
Ángela Mourelle-Insua, Iván Lavandera,* and Vicente Gotor-Fernández*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Deep eutectic solvents (DES) represent nowadays a sustainable use of chemical, electrochemical or photochemical
5
alternative to traditional organic solvents in (bio)transformations. transformations, or the use of cheaper synthesised
6
Herein, the use of a solvent composed by an aqueous buffer and nicotinamide cofactor mimetics in stoichiometric amounts.
choline chloride: glucose (1.5:1 mol/mol) is proposed, a natural Deep eutectic solvents (DES) have recently appeared as a new
7
DES (NADES) serving as both cosolvent and efficient system to generation of ionic liquids (ILs). A DES is typically composed
recycle the nicotinamide cofactor. Thus, glucose from NADES by a quaternary ammonium salt acting as hydrogen bond
served as co-substrate required for several alcohol acceptor (HBA, i.e. choline chloride), and a hydrogen bond
dehydrogenases to reduce different prochiral ketones, and also donor (HBD, such as glycerol, urea or a sugar), so the
helped to solubilise the organic compounds to develop effective intermolecular interactions between its components provide
biotransformations at higher substrate concentrations.
specific properties for these neoteric solvents. Their
straightforward preparation by simply mixing a HBA and a HBD
to form a liquid, represents an excellent waste-free process
providing a significant advantage in comparison with the
preparation of traditional ILs. Moreover, the possibility of
utilising natural and degradable compounds is also highly
valuable from an environmental point of view.
The application of oxidoreductases (EC.1) in organic synthesis
has gained maturity in the last decades due to their high levels
of activity and selectivity displayed in redox processes.
1
Particularly alcohol dehydrogenases (ADHs, EC.1.1.1.x)
represent a sustainable alternative to non-enzymatic methods
for the production of optically active alcohols from racemic
In this context, natural deep eutectic solvents (NADES) are
defined as a mixture of two or more compounds that are
generally plant based primary metabolites (organic acids,
sugars, alcohols, amines and amino acids), that interact via
intermolecular forces and liquefy if combined in specific molar
2
and prochiral carbonylic compounds. This class of redox
catalyst requires the use of a cofactor, β-nicotinamide adenine
+
dinucleotide, which exists in a phosphorylated (NADP ) and
+
non-phosphorylated (NAD ) oxidised form, or in their reduced
versions [NAD(P)H]. These cofactors can mediate as electron
acceptors/donors in either oxidation or reduction processes.
Since the use of cofactors in stoichiometric amounts is
hampered due to inhibition effects and economic hurdles, the
employment of cofactor recycling systems is compulsory for
the development of efficient and economic feasible redox
transformations.3 For the case of reductive reactions, this
normally implies the coupling of a second enzymatic reaction
8
ratios. The NADES concept dates from 2011 and it created
great expectations in the context of Green Chemistry as they
are less toxic and more environmentally friendly than ionic
liquids (ILs) and traditional metal-based DES.
Interestingly, the use of (NA)DES as green (co)solvents in
9
enzymatic transformations has rapidly gained attention,
10
including their application to redox processes. In this context,
the development of bioreductions using ADHs appears as a
robust technique for the asymmetric synthesis of alcohols
using aqueous-(NA)DES mixtures, where mainly choline
4
typically mediated by glucose dehydrogenase (GDH) or
formate dehydrogenase (FDH) at expenses of a sacrificial
cosubstrate, glucose or formate, respectively, in the so-called
coupled-enzyme approach. Other methodologies involve the
11
chloride: glycerol (ChCl:Gly) has been employed as cosolvent.
In some cases, other HBDs have also been used, the choice of
glucose being scarcely reported and in all cases for whole cell-
1
2
mediated biotransformations. Herein, we have focused on
Organic and Inorganic Chemistry Department, University of Oviedo, Avenida Julián
Clavería 8, 33006 Oviedo (Spain). E-mail: lavanderaivan@uniovi.es (IL);
the development of efficient global reduction processes by
vicgotfer@uniovi.es (VGF); Fax: +34 985103446; Phone: +34 985103452 (IL), +34 combining an overexpressed ADH for the reduction of
9
†
85103454 (VGF).
prochiral ketones accomplished with an appropriate cofactor
recycling system, considering in this case glucose
Electronic Supplementary Information (ESI) available: Bioreduction experiments,
1
13
analytics, copies of HPLC chiral analyses, and H, C and DEPT NMR spectra for
described organic compounds are included. See DOI: 10.1039/x0xx00000x
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Journal Name
dehydrogenase (GDH), which allows the cofactor reduction by (ChCl) and glucose (1.5:1 mol/mol) was added intoVietwheArtriceleaOctniloinne
transforming D-glucose into D-glucono-1,5-lactone that later medium (50 mM Tris·HCl pH 7.5, 1 mM NADDOPIH: 1)0..1F0in3a9l/lCy9, G1C00m03g18oEf
spontaneously hydrolyses in the reaction medium towards the the lyophilised ADH were resuspended and the reactions were
formation of D-gluconic acid. For that reason, we decided to incubated at 30 °C for 24 h. The results from the bioreduction
investigate the use of D-glucose as HBD in a designer NADES experiments are shown in Table 1.
with an efficient solution for: (a) Better solubility of the
Table 1 Bioreduction of ketones using ADHs in aqueous medium and 10% v/v of NADES
lipophilic substrates; and (b) Suitable cofactor regeneration
ChCl:Glu (1.5:1 mol/mol) as cosolvent.a
system (Scheme 1).
ADH
O
OH
*
O
ADH
OH
*
NADPH (1 mM), GDH
R1
R2
50 mM Tris·HCl pH 7.5
0% v/v ChCl:Glu (1.5:1 mol/mol)
GDH (3 units)
R1
R2
R1
R2
R1
R2
1
1a-c
2a-c
(
25 mM)
NAD(P)+
NAD(P)H
30 ºC, 24 h, 250 rpm
DES
OH
O
Conv. (%)b
ee (%)b
Entry
ADH
Lb
TeS
T
Sy
Ras
Substrate
OH
O
HO
1
2
3
4
5
Acetophenone (1a)
2-Octanone (1b)
2-Octanone (1b)
Propiophenone (1c)
Propiophenone (1c)
>99
86
99
81
>99
>99 (R)
92 (S)
>99 (S)
>99 (S)
>99 (S)
HO
HO
HO
OH
GDH
HO
HO
O
N
OH
Cl
Scheme 1 Use of a designer deep eutectic solvent formed by D-glucose and ChCl to
recycle the nicotinamide cofactor [NAD(P)H] in alcohol dehydrogenase-catalysed
processes.
a
See the ESI file for detailed reaction conditions and protocols. b Conversion and
enantiomeric excess values were determined by GC analysis. The absolute
configuration of the alcohol appears in brackets.
Gratifyingly, complete conversions and selectivities were attained
for LbADH, ADH-T and RasADH in the bioreduction of
acetophenone, 2-octanone and propiophenone, respectively
For this study, commercially available GDH-105 from Codexis Inc.
was selected for cofactor recycling, and the behaviour of different
made in house overexpressed NADPH-dependent alcohol
(entries 1, 3 and 5), while TeSADH (entry 2) and SyADH (entry 4)
dehydrogenases (ADHs) was studied. We used the (R)-selective one
from Lactobacillus brevis (LbADH)13 and the (S)-selective ADHs from
also led to high conversions and good to excellent enantiomeric
excess. As a result of their remarkable performances, we decided to
delve into bioreductions catalysed by LbADH, ADH-T and RasADH
using this designer NADES as both co-solvent and co-substrate.
As one could argue regarding the integrity of the NADES in aqueous
medium at low concentrations, we decided to increase its
concentration in order to study this effect. Edler and co-workers
recently showed that using DES in water at >50% (w/w)
1
4
Thermoanaerobacter ethanolicus (TeSADH), Thermoanaerobacter
1
5
16
sp. (ADH-T), Sphingobium yanoikuyae (SyADH) and Ralstonia sp.
(
1
7
RasADH). To check the enzyme activity in the presence of NADES,
the model substrate for each ADH was added in 25 mM
concentration, ideal conditions for this class of redox enzymes, and
commercial GDH-105 was used as coupled-enzyme (3 units). From
ChCl:Glu mixtures, the 1.5:1 mol/mol was selected in order to have
a significant amount of glucose for cofactor recycling purposes
without the detriment of using a highly dense and viscous NADES at
considerable glucose concentrations, which would make it difficult
1
9
concentrations, the nanostructure of the eutectic solvent is
perfectly retained (see also NMR experiments in the ESI, Figures S2-
S8). Therefore, we increased the proportion of NADES employed in
2
0
1
8
the biotransformation up to 90% v/v (see Fig. 1).
to handle. 10% v/v of the NADES formed by choline chloride
Fig. 1 Effect in activity and selectivity using different amounts of NADES ChCl:Glu (1.5:1 mol/mol) in the bioreduction of acetophenone (LbADH), 2-octanone (ADH-T) and
propiophenone (RasADH). Numerical values are given in the ESI.
2
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Fig. 2 Effect in activity and selectivity using different amounts of NADES ChCl:Glu (1.5:1 mol/mol) at variable substrate concentrations in the bioreduction of acetophenone
LbADH), 2-octanone (ADH-T) and propiophenone (RasADH). Numerical values are given in the ESI.
(
Gratifyingly, LbADH and ADH-T revealed perfect results in terms of activity of ADH-T was worse in comparison when using 10% v/v of
conversion and enantioselectivity at high NADES contents. On the NADES, LbADH and RasADH acted in a similar manner than in the
one hand, LbADH catalyzed quantitatively the bioreduction of reaction with 10% NADES.
acetophenone even at 60% v/v NADES concentration, maintaining The best substrate concentration vs conversion ratio for each
complete selectivity even at 80% v/v of NADES (>99% ee). On the enzyme was selected in order to compare the effect of our system
other hand, RasADH led to perfect conversions until 70% v/v of regarding the standard glucose/GDH system typically employed
NADES in the bioreduction of propiophenone, but a drop in the (Table 2). Using the same amount of glucose for all the
selectivity was observed when using a proportion equal or higher bioreductions, the NADES/GDH system was superior (even entries)
than 40% v/v of NADES (93% ee). Interestingly, this surprising to the biotransformations performed by just adding glucose/GDH
change in the stereoselectivity has been reported for several (odd entries), especially for the bioreduction of acetophenone with
authors when using different alcohol dehydrogenases (e.g., LbADH (entries 1 and 2). While this effect remains unclear, there
1
1b
RasADH), and it is usually associated with the inhibition of other are reports that have described that choline chloride can exert a
2
1
biocatalytic activities present in the enzyme preparation or due to stabilizing effect in the structure of biomolecules such proteins or
1
0
22
protein conformational changes.
DNA.
To show the applicability of this methodology, the use of higher
substrate concentrations was explored, ranging from 30 to 200 mM
Table 2 Bioreduction of ketones using ADHs with glucose as co-substrate in pure buffer
or using a 10% v/v ChCl:Glu mixture as cosolvent at 30 °C and 250 rpm for 24 h.a
(
Fig. 2) under similar reaction conditions to those previously
reported at 25 mM. First of all, 10% v/v of NADES was employed
Fig. 2A) and different trends were observed. On the one hand,
ADH
O
OH
*
NADPH (1 mM), GDH
(
R1
R2
R1
R2
50 mM Tris·HCl pH 7.5
ADH-T and RasADH catalysed the complete reductions of 2-
octanone and propiophenone up to 50 mM, yielding both alcohols
in enantiopure form. ADH-T displayed lower activities at 75 mM
substrate concentration (75% conversion) with a slight decrease in
the selectivity (96-98% ee at 75-200 mM), while RasADH led to 83%
conversion at 100 mM, to later significantly decrease the activity
although the selectivity was optimum in all cases. On the other
hand, LbADH was capable of transforming up to 100 mM of
acetophenone into enantiopure (R)-1-phenylethanol with complete
conversion.
0
-10% v/v NADES ChCl:Glu (1.5:1)
1a-c
50-100 mM)
2a-c
30 ºC, 24 h, 250 rpm
(
Entry
NADES (%)
ADH-Substrateb
Conv. (%)c
ee (%)c
>99 (R)
>99 (R)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
1
2
3
4
5
6
0
10
0
10
0
Lb and 1a (100 mM)
Lb and 1a (100 mM)
T and 1b (50 mM)
T and 1b (50 mM)
Ras and 1c (100 mM)
Ras and 1c (100 mM)
59
>99
89
98
61
10
83
Having in mind that glucose is employed as co-substrate, so its
concentrations decreased with the advance of the reaction, higher
amounts of NADES were employed (30% v/v of NADES) at different
a
See the ESI file for detailed reaction conditions and protocols. b The ketone
concentration is shown in brackets, while the glucose concentration is 240 mM.
c
Conversion and enantiomeric excess values were determined by GC analysis.
ketone concentrations (25-200 mM, Fig. 2B). Remarkably, the three The absolute configuration of the alcohol appears in brackets.
ADHs worked with excellent levels of selectivity, and although the
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Selected bioreduction processes were monitored within the time, later spontaneously hydrolyses to D-gluconic acid aVsiewbyAr-tpicrleoOdnulcinte,
trying to find optimal conditions for the selective bioreduction of decreasing the pH of our aqueous system (TDrOisI:·H10C.l105309/mCM9GCp0H0731.58)E.
the ketones (Table 3). 30% v/v NADES, 25 mM ketone In order to test the influence of these two parameters in the
concentrations and 30 °C were fixed, achieving full conversions for enzyme activity, firstly pentane was used instead of EtOAc to
each substrate at 1 h (acetophenone, LbADH, entry 2), 1.5 h (2- extract the product (Fig. 4, blue bars), and later the Tris·HCl buffer
octanone, ADH-T, entry 5) or 20 min (propiophenone, RasADH, 200 mM pH 7.5 was also employed instead of the previous 50 mM
entry 8).
buffer (Fig. 4, red bars) in order to see if a higher buffer
Also, the recyclability of the enzyme and NADES (30% v/v) was concentration could balance out the production of D-gluconic acid
subjected to study (Fig. 3). After the first transformation was without altering the pH of the buffer. The results are shown in Fig.
completed, the reaction mixture was extracted in high yields with 4, observing significant benefits when using pentane and the 200
EtOAc (upper phase), showing full conversions and excellent mM Tris HCl buffer.
selectivities, and the aqueous medium formed by the NADES and Interestingly, the use of pentane already led to better results
the enzymes (ADH and GDH, bottom phase) was employed for allowing a successful first recycling with LbADH (Fig. 4 left, 97%
running
a second biotransformation by simply adding the conversion) and RasADH (Fig. 4 right, 99% conversion). In order to
corresponding ketone 1a-c. Unfortunately, a significant activity loss explore the possible enzyme deactivation due to the extracting
was observed (10-56% conversion), which was dramatic for LbADH solvent, additional experiments (Fig. 4, green bars) were carried out
(
Fig. 3, left) and ADH-T (Fig. 3, middle).
by filtering the ADH after the bioreduction, to later extract the
reaction medium and perform the next reuse. In this case only
slight improvements were found for LbADH (Fig. 4, left), while not
significant changes for the recycling of ADH-T (Fig. 4, middle) and
RasADH (Fig. 4, right). When the pH value of the reaction medium
after a biotransformation was measured, it was found 3.5 for those
Table 3 Conversions vs time of alcohol dehydrogenase-catalysed bioreductions using
3
0% v/v NADES ChCl:Glu (1.5:1).a
ADH
O
OH
*
NADPH (1 mM), GDH
R1
R2
R1
R2
.
50 mM Tris·HCl pH 7.5
performed in the Tris HCl buffer 50 mM, while a value around 4.9-
3
0% v/v NADES ChCl:Glu (1.5:1)
1
a-c
2a-c
5.0 was attained in the ones carried out at a 200 mM buffer
concentration due to the release of D-gluconic acid. For that reason,
pH was readjusted up to 7.5 before each recycling experiment after
enzyme filtration and extraction with pentane (Fig. 4, purple bars),
finding in this manner a great improvement, especially when using
LbADH (98% conversion and 98% ee after 5 cycles, Fig. 4 left) and
RasADH (98% conversion and >99 ee after 8 cycles, Fig. 4 right and
ESI).
Finally, in order to demonstrate the applicability of the method we
set up some semi-preparative biotransformations (100 mg of
ketone) finding results in accordance with those obtained at
analytical scale. Full conversions into the enantiopure alcohols were
reached, producing (R)-1-phenylethanol in 78% isolated yield with
LbADH (150 mg of the lyophilised preparation and 100 mM ketone
concentration), (S)-2-octanol in 85% yield using ADH-T (350 mg of
the lyophilised preparation, 50 mM) and (S)-1-phenyl-1-propanol in
30 ºC, t, 250 rpm
(
25 mM)
Entry
ADH
Lb
Lb
T
T
Substrate
t (min)
30
60
30
60
90
10
15
20
Conv. (%)b
70
ee (%)b
1
2
3
4
5
6
7
8
1a
1a
1b
1b
1b
1c
1c
1c
>99 (R)
>99 (R)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99
82
95
>99
97
98
T
Ras
Ras
Ras
>99
a
See the ESI file for detailed reaction conditions and protocols. b Conversion and
enantiomeric excess values were determined by GC analysis. The absolute
configuration of the alcohol appears in brackets.
There are two possible reasons to explain this inactivation effect.
On the one hand, the use of a great amount of an organic solvent
such as EtOAc could lead to this effect. On the other hand, D-
glucono-1,5-lactone is produced through the glucose oxidation that
8
9% yield with RasADH (150 mg of the lyophilised preparation, 100
mM), after extractions with ethyl acetate.
Fig. 3 Recycling of the enzyme and 50mM buffer-NADES (30% v/v) mixture after extraction with EtOAc in the bioreductions of acetophenone with LbADH
left), 2-octanone with ADH-T (middle) and propiophenone with RasADH (right). Numerical values are given in the ESI.
(
4
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Fig. 4 Recycling of the enzyme and NADES (30% v/v) after extraction with pentane in the bioreductions of acetophenone with LbADH (left), 2-octanone with
ADH-T (middle) and propiophenone with RasADH (right): A) Blue bars are reactions carried out in 50 mM buffer, extracted with pentane and reusing the
NADES-buffer plus the enzyme; B) Red bars are reactions carried out in 200 mM buffer, extracted with pentane and reusing the NADES-buffer plus the
enzyme; C) Green bars are reactions carried out in 200 mM buffer, filtering the enzyme, extracting with pentane and reusing the NADES-buffer plus the
enzyme; D) Purple bars are reactions carried out in 200 mM buffer, filtering the enzyme, extracting with pentane, readjusting the pH of the NADES-buffer
mixture up to 7.5 and reusing the NADES-buffer plus the enzyme. Numerical values are given in the ESI.
For these semi-preparative transformations, we performed a simple
Acknowledgements
Financial support from the Spanish Ministry of Economy and
Competitiveness (MEC, Project CTQ2016-75752-R) is gratefully
acknowledged. A.M.-I. thanks MEC for a predoctoral fellowship
inside the FPI program.
quantification of the E-factor23 to obtain an overview of the
2
4
environmental impact of this methodology. Hence, the EATOS tool
was used focusing on the relevance of the reaction conditions
regarding the reagents, catalysts and solvents employed (excluding
water), and taking into account the waste generated. As can be
seen in the ESI (Figure S1), the values obtained were between 236-
2
86. While these numbers are still high, it can be seen that the
Notes and references
highest percentage of these numbers come from the solvents
employed, in particular from EtOAc used to extract the final
products. Since at big scale the recycling of organic solvents is a
common applied technique, we are sure that these numbers could
be further optimised.
1
a) D. Monti, G. Ottolina, G. Carrea and S. Riva, Chem. Rev.,
011, 111, 4111; b) Synthetic Methods for Biologically Active
2
Molecules. Exploring the Potential of Bioreductions, Ed. E.
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Overall, a ChCl:Glu mixture has been used as a designer natural DES
applied to ketone bioreduction transformations using five different
ADHs. The combination of an aqueous buffer system with the
NADES in up to 50% v/v ratio has provided two main advantages.
On one hand, the presence of glucose provides the co-substrate for
the GDH-catalysed reaction for the nicotinamide cofactor recycling.
On the other hand, the bioreductions were run at higher substrate
concentrations in comparison with the buffer system employing
glucose/GDH, and the development of practical protocols in terms
of excellent conversions were possible in up to 100 mM
concentration with excellent selectivities. After optimisation of the
reaction conditions, the bioreductions were carried out in semi-
preparative scale finding that the use of pentane as extracting
organic solvent presented great advantages for enzyme recycling in
comparison with ethyl acetate, traditional solvent used in these
work-up procedures, as pentane preserve better from enzyme
inactivation. It is also worth mentioning that the use of
concentrated buffers (i.e. 200 mM Tris HCl buffer) highly improved
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is formed as co-product in the cofactor recycling reaction,
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