Rapid, Heterogeneous Biocatalytic Hydrogenation and Deuteration in a Continuous Flow Reactor

Article abstract of DOI:10.1002/cctc.202000161

The high selectivity of biocatalysis offers a valuable method for greener, more efficient production of enantiopure molecules. Operating immobilised enzymes in flow reactors can improve the productivity and handling of biocatalysts, and using H₂ gas to drive redox enzymes bridges the gap to more traditional metal-catalysed hydrogenation chemistry. Herein, we describe examples of H₂-driven heterogeneous biocatalysis in flow employing enzymes immobilised on a carbon nanotube column, achieving near-quantitative conversion in <5 min residence time. Cofactor recycling is carried out in-situ using H₂ gas as a clean reductant, in a completely atom-efficient process. The flow system is demonstrated for cofactor conversion, reductive amination and ketone reduction, and then extended to biocatalytic deuteration for the selective production of isotopically labelled chemicals.

Full text of DOI:10.1002/cctc.202000161

The European Society Journal for Catalysis
Supported by
Accepted Article
Title: Rapid, Heterogeneous Biocatalytic Hydrogenation and
Deuteration in a Continuous Flow Reactor
Authors: Lisa Alice Thompson, Jack Steven Rowbotham, Jake H
Nicholson, Miguel A Ramirez, Ceren Zor, Holly A Reeve,
Nicole Grobert, and Kylie Alison Vincent
This manuscript has been accepted after peer review and appears as an
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Link to VoR: https://doi.org/10.1002/cctc.202000161
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1/2020

ChemCatChem
10.1002/cctc.202000161
FULL PAPER
Rapid, Heterogeneous Biocatalytic Hydrogenation and
Deuteration in a Continuous Flow Reactor
[a]
[a]
[a]
[a]
[b]
Lisa A. Thompson, Jack S. Rowbotham, Jake H. Nicholson, Miguel A. Ramirez, Ceren Zor,
[
a]
[b]
[a]
Holly A. Reeve, Nicole Grobert and Kylie A. Vincent*
[
[
a]
b]
Dr. L. A. Thompson, Dr. J. S. Rowbotham, Mr. J. H. Nicholson, Dr. M. A. Ramirez, Dr. H. A. Reeve, Prof. K. A. Vincent
Department of Chemistry
University of Oxford
Inorganic Chemistry Laboratory, South Parks Road, Oxford, OX1 3QR, UK
E-mail: kylie.vincent@chem.ox.ac.uk
Ms. C. Zor, Prof. N. Grobert
Department of Materials
University of Oxford
Parks Road, Oxford, OX1 3PH, UK
Supporting information for this article is given via a link at the end of the document.
Abstract: The high selectivity of biocatalysis offers a valuable method
for greener, more efficient production of enantiopure molecules.
Operating immobilised enzymes in flow reactors can improve the
These methods employ additional enzymes and sacrificial
substrates, such as glucose dehydrogenase and glucose or
alcohol dehydrogenase and isopropanol, generating significant
quantities of carbon-based waste. Despite a rapidly growing
interest in continuous processing in chemical production, the
complexity of biocatalytic systems, together with difficulties in
enzyme immobilisation, have hindered translation of biocatalysis
into flow operation.
productivity and handling of biocatalysts, and using H
redox enzymes bridges the gap to more traditional metal-catalysed
hydrogenation chemistry. Herein, we describe examples of H -driven
2
gas to drive
2
heterogeneous biocatalysis in flow employing enzymes immobilised
on a carbon nanotube column, achieving near-quantitative conversion
in <5 min residence time. Cofactor recycling is carried out in-situ using
Continuous flow reactors have been widely used for organic
synthesis over the past few decades,[2,3] and are increasingly
being explored for biocatalysis.[4,5] Flow offers a number of
advantages over batch,[6] including increased productivity,
improved safety when handling toxic reagents or explosive gases,
2
H gas as a clean reductant, in a completely atom-efficient process.
The flow system is demonstrated for cofactor conversion, reductive
amination and ketone reduction, and then extended to biocatalytic
deuteration for the selective production of isotopically labelled
chemicals.
[
7–10]
such as H
monitoring.[
2
,
and ease of automation and in-line reaction
Another advantage of flow chemistry is the ability
11–14]
to retain catalysts within a column-type reactor, whilst pumping
the reaction solution through the solid, allowing for easy
separation, recovery and potential re-use of the catalyst, as well
as increased contact between the solution and solid catalyst.[
Immobilisation of biocatalysts often improves stability and
tolerance to organic solvents and elevated temperatures.[18,19] The
Introduction
15–
Typical chemical approaches for the reduction of unsaturated
bonds in organic molecules rely on hydrogenation using
molecular or supported nanoparticle metal catalysts, or hydride
17]
constant removal of reaction solution in flow can also prevent or
minimise any product inhibition that may be experienced by the
transfer from a donor such as NaBH
4
. Direct hydrogenations with
H
2
benefit from atom-efficiency, but often suffer from harsh
biocatalyst. As a result, there is an increasing number of
reaction conditions, poor selectivity and contamination of the
organic product with toxic metals. Biocatalysis is increasingly
considered as a greener alternative for sustainable C=X bond
reductions due to the high chemo-, regio- and stereo- selectivity
of enzymes under mild reaction conditions.[1] However,
challenges still remain which prevent the widespread adoption of
redox biocatalysis. Key barriers include the (often) high
production cost and limited stability of biocatalysts, as well as the
requirement of many redox enzymes for expensive cofactors. A
large number of biocatalytic C=X bond reductions occur via
delivery of hydride from the reduced form of a nicotinamide
cofactor (NADH or NADPH), generating the oxidised cofactor,
[20]
examples of immobilised biocatalysts operated in flow,
cofactor recycling provided by traditional methods.
with
[21–23]
However,
there is currently no standard method for handling immobilised
redox biocatalysts in flow, and so wider adoption outside the
biotechnology community is limited. Therefore, a biocatalytic
approach that more closely resembles the heterogeneous
chemical methods already in widespread use in flow reactors
could facilitate uptake of redox biocatalysis in flow.
To
capture
the
advantages
of
heterogeneous
hydrogenations and biocatalysis for selective C=X bond
reductions, we have developed a biocatalytic NADH recycling
gas as a clean reductant (Figure 1).[24,25]
system that uses only H
2
+
NAD(P) . The high cost of these cofactors means that it is not
The enzymes are immobilised on a carbon support by simple
adsorption, requiring no covalent attachment chemistry and no
modification of the enzymes. The cofactor recycling enzymes can
feasible to add them in stoichiometric quantity, and so cofactor
recycling methods have been developed and are widely used.
1
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ChemCatChem
10.1002/cctc.202000161
FULL PAPER
+
be coupled to a range of NADH-dependent enzymes, selective
In this reaction, the liquid feed was comprised of 2 mM NAD in
biotransformations are driven by H
The NADH-dependent C=X bond reductase enzyme can also be
2
, as indirect hydrogenations.
Tris-HCl buffer (50 mM, pH 8.0). The liquid flow rate was 32 µL
-1
-1
min and the gas flow rate was 100 µL min . The reaction solution
was collected as 2 x reactor volume (RV) fractions, at the reactor
outlet and analysed by UV-Vis spectroscopy. Quantitative
co-immobilised onto the support material, creating
heterogeneous biocatalytic cascade.
We recently described initial results using
biocatalytic systems supported in a simple flow reactor. This
combines the advantages of supported enzymes in flow, with H
a
fully
+
H
2
-driven
conversion of NAD to NADH was achieved in only a 2.2 min tRes
[
26]
and was maintained for 40 RV (Figure 2B). This corresponded to
1544 enzyme turnovers, with a turnover frequency (TOF) of 18
2
min , and a space-time yield (STY) of 38.7 gProduct L-1 h-1. Good
reproducibility was also achieved in the flow reactor, see SI.
The high conversion and short tRes in a single-pass
reaction demonstrates a large improvement over the previous
biocatalytic reactor set-up and enzyme system we described,
-1
gas for greener cofactor recycling, to overcome some of the key
challenges of biocatalysis. This set-up employed hydrogenase /
+
[27]
2
NAD -reductase as the H -driven cofactor recycling system, co-
immobilised within a quartz column coated with multi-wall carbon
nanotubes using chemical vapour deposition techniques.[26]
2
However, the use of a single H -saturated liquid feed meant that
2
owing to the increased H availability.
H
2
availability was limiting in the reactor set-up, and these initial
studies required re-circulation of the reaction solution through the
reactor overnight (semi-continuous flow) to produce modest
conversions. For example, we reported 67% conversion of NAD+
to NADH in 6.5 h of re-circulation through the reactor. When
combined with NADH-dependent dehydrogenases, this system
gave enantiopure chiral products in 30 – 90% conversion after
overnight re-circulation.
(
A)
Here, we demonstrate significant improvements to the
heterogeneous biocatalytic flow reactor set-up, optimising the H
2
availability so that we achieve high conversions in a single-pass
with short residence times (tRes). We demonstrate this for a
number of biotransformations, using soluble hydrogenase (SH)
(
B)
100
40
from Ralstonia eutropha as the H
and various NADH-dependent reductase enzymes. We further
extend this approach to include biocatalytic deuteration in flow.
2
-driven NADH recycling enzyme,
35
80
60
40
20
0
30
25
20
15
10
5
0
3
00
400
500
Wavelength / nm
0.0
0.5
1.0
1.5
Time / h
Figure 2. (A) Carbon nanotube column flow reactor for H
cofactor conversion using soluble hydrogenase (SH); (B) Cofactor conversion
2
-driven biocatalytic
2
Figure 1. Heterogeneous biocatalytic H -driven cofactor recycling: A cofactor
recycling system and an NADH-dependent reductase enzyme are co-
immobilised on a carbon support to carry out a selective biotransformation, so
(
■) over time in the flow reactor, showing cumulative reactor volumes (▲).
+
Reaction conditions: SH (12 U), 2 mM NAD , 50 mM Tris-HCl buffer, pH 8.0,
2
that the biocatalytic C=X bond reduction is driven by H .
H
2(g), 2.2 min tRes, gas:liquid ratio = 3:1. Inset shows UV-Vis spectrum for
NADH product.
Results and Discussion
The reaction was continued up to 200 RV (7.3 h), during
Continuous H
2
-driven Biocatalytic Cofactor Reduction
which the SH remained active, however, the conversion
decreased to 60%. Over this whole 200 RV operating time the
total enzyme turnover number (TTN) was 6077 and the TOF was
Experiments in this study employed the single-walled carbon
nanotube lined quartz tubes that we have previously used for
biocatalysis in flow.[26] To improve H
the set-up was modified to include a direct H
A). The separate liquid and gas feeds were combined at a Y-
availability within the reactor, 11 min-1 by the end of the reaction. Enzyme turnover numbers
2
2
gas feed (Figure
and frequencies were calculated assuming all of the SH added to
the flow column had adsorbed onto the carbon nanotubes,
therefore it is likely that these values are an under-estimate of the
activity of the immobilised enzyme sample. The loss of SH activity
in the flow reactor was probed further through repeated flow
experiments with different tRes (Figure 3), and it was observed
that the decrease in conversion occurred after 1200-1500 SH
turnovers, regardless of the total time-on-stream.
2
shaped connector prior to entering the column reactor, and the
flow rates adjusted to give a gas:liquid feed ratio of approximately
3:1.
This modified reactor set-up was first applied to develop a
single-pass flow reaction for H -driven NADH production using SH
immobilised in the carbon nanotube column reactor (Figure 2A).
2
2
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ChemCatChem
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-
1
This demonstrated the improved reactor set-up as a viable
method for rapid H -driven turnover of cofactor, with the potential
to provide efficient cofactor recycling when coupled to other
NADH-dependent enzymes.
(Figure 4B). The SH TOF was 79 min in the high activity region,
which was higher than the TOF observed for the cofactor
conversion experiments described in the previous section. In the
2
+
SH-only cofactor reduction experiments, 2 mM NAD was used.
Whereas 10 mM substrate 1 was used in these reductive
+
amination studies, with 1 mM NAD . Therefore, the SH had to turn
over the cofactor molecules more times to be able to supply
sufficient NADH (10 mM) for the production of 2. Whilst Entry 3
generated a slightly lower conversion (90%), the shorter tRes
provided a higher STY of 27.3 gProduct L-1 h-1 as more material was
processed per unit time. These results represent
a vast
improvement on previous results, in which overnight re-circulation
generated just 40% conversion for the same reaction.[26]
(
A)
(B)
100
80
60
40
20
0
0
1
2
3
4
5
Time / h
Figure 3. Comparison of multiple flow experiments showing SH activity loss
+
during NAD conversion to NADH for: 10 min tRes (▲), 2.2 min tRes (●), and 1
min tRes (■). Plotted in terms of (A) Reactor volumes and (B) Reaction time.
Reaction conditions: SH (10 U), 2 mM NAD , 50 mM Tris-HCl buffer, pH 8.0,
2
Figure 4. (A) H -driven biocatalytic reductive amination in flow using alanine
+
dehydrogenase (AlaDH) and soluble hydrogenase (SH). (B) Conversion of
pyruvate to alanine using AlaDH and SH (●), overlaid with cofactor reduction
when using only SH (■), showing a consistent decrease in conversion. Reaction
H
2(g), gas:liquid ratio = 3:1. TN = enzyme turnover number.
+
conditions: SH, AlaDH, 10 mM sodium pyruvate, 1 mM NAD , 50 mM Tris-HCl
buffer, pH 8.0, H2(g), gas:liquid ratio = 3:1, 5 min tRes.
Continuous H
The flow reactor was next tested for the biocatalytic reductive
amination of pyruvate 1 with in-situ H -driven cofactor recycling
Figure 4A). For this step, SH and NADH-dependent alanine
2
-driven Biocatalytic Reductive Amination
2
2
Table 1. Biocatalytic production of alanine 2 in flow with H -driven cofactor
recycling.
(
dehydrogenase (AlaDH) were co-immobilised within the column
Entry[a]
tRes / min
Cofactor / mol%
Conversion
reactor. The liquid feed contained 10 mM sodium pyruvate, 100
[b]
/
%
+
mM NH
4
Cl as the amine donor, and different amounts of NAD , in
1
2
3
4
15
5
10
10
10
1
>99
>99
90
Tris-HCl buffer (50 mM, pH 8.0). Flow rates were adjusted to
achieve the desired tRes through the reactor, whilst maintaining
the 3:1 gas:liquid ratio. The reaction solution was collected as 8-
1
0 combined RV samples at the reactor outlet and the conversion
2.2
5
1
of pyruvate 1 to alanine 2 was monitored by H NMR spectroscopy
23
(
Table 1). Using 10 mol% cofactor, high conversions were
achieved for 15, 5 and 2.2 min tRes, respectively (Entries 1-3).
Only 23% conversion was achieved on decreasing the cofactor to
[
a] Reaction conditions: SH (10U), AlaDH (10U), 10 mM sodium pyruvate, 100
mM NH Cl, 50 mM Tris-HCl (pH 8.0), gas:liquid ratio = 3:1, RV = 288 µL; [b]
Determined by H NMR spectroscopy.
4
1
1
mol%, for a 5 min tRes (Entry 4). For Entry 2, high conversion
was maintained across 24 RV, providing a STY of 13.3 gProduct L-1
-
1
h . After this a decrease in conversion was observed that was
consistent with the observed drop in activity of the SH over time
3
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ChemCatChem
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+
conversion of 2 mM NAD 7 to the deuterated reduced form [4S-
2H]-NADH 8 was achieved in 2.2 min in the flow reactor, and with
>99% isotopic selectivity. This was consistent with the conversion
achieved for the production of non-deuterated NADH in the flow
reactor, described above, demonstrating straightforward
transferal of the flow protocol to generate isotopically labelled
products.
2
Continuous H -driven Biocatalytic Ketone Reduction
An NADH-dependent alcohol dehydrogenase (ADH105, Johnson
Matthey) was also implemented in the flow reactor for the
selective reduction of 4’-chloroacetophenone 3 to (S)-1-(4-
chlorophenyl)ethanol (S)-4 (Scheme 1A), in combination with SH
2
for H -driven cofactor recycling. Using the carbon nanotube
2
column reactor this provided 90% conversion of 10 mM 3 to (S)-4
in 2.2 min tRes, with 10 mol% cofactor. However, a decrease in
conversion was observed after only 8 RV (to 1% after 100 RV, SI).
This may have been due to poorer adsorption of the ADH on to
the carbon nanotubes, leading to leaching of the enzyme from the
reactor. Further development is underway to improve the stability
of this biocatalytic system, including the use of different carbon
supports for ADH105 immobilisation in flow.
Having demonstrated the formation of [4S- H]-NADH in the
flow reaction setup, the system could then be extended to
produce labelled [3R- H]-3-quinuclidinol 9 from 5 by including the
AtQR (Scheme 2B). Operating under buffered H O, >99%
conversion from 5 to 9 was achieved, with ≥95% deuterium
incorporation at C3, in a 5 min tRes. Additional H incorporation
was also observed on C2 of the product, owing to non-enzymatic
exchange of the enolisable protons on the substrate with the
solvent (see SI). After an initial period of high conversion, a
subsequent decrease in the production of the alcohol was
observed from 2687 SH turnovers onwards, consistent with the
loss of activity of the SH discussed earlier. However an SH TOF
of 77 min-1 was achieved in the initial period, and a TTN of 10206
was achieved over the whole operating time, indicating that the
SH activity was not negatively impacted by switching to the
deuterated buffer system. Upon further development, the process
described here could prove to be a valuable route to selectively
deuterated pharmaceutical building blocks in continuous flow,
2
2
2
2
made accessible by the straight-forward translation of reactions
2
into H
2
O operating conditions.
Scheme 1. H
2
-driven biocatalytic ketone reduction in flow. (A) Conversion of 3
using ADH105 and SH; (B) Conversion of 5 using AtQR and SH. (SH = soluble
hydrogenase, ADH105
reductase).
= alcohol dehydrogenase, AtQR = quinuclidinone
An additional ketone substrate was next explored using the
quinuclidinone reductase from Agrobacterium tumefaciens
(
AtQR) for the reduction of 3-quinuclidinone 5 to (R)-3-
quinuclidinol 6 (Scheme 1B). The product, 6, is an important
component of a number of pharmaceutical compounds, and so
methods to selectively produce the enantiomerically pure alcohol
are a valuable synthetic tool.[28,29] For this substrate, the flow
system was again tested with both AtQR and SH co-immobilised
within the carbon nanotube column reactor, which gave
Scheme 2. (A) H
in flow; (B) H
*
2
-driven biocatalytic deuteration of NAD+ 7 to [4S- H]-NADH 8
2
quantitative conversion of 5 mM substrate to (R)-6 in a 5 min tRes.
2
This provided an SH TOF of 60 min-1 and STY of 9.8 gProduct L-1 h-
2
-driven biocatalytic production of [3R- H]-3-quinuclidinol 9 in flow.
Non-enzymatic 2H incorporation at C2 was also detected (see SI). (SH =
1,
2
and demonstrated the potential of H -driven biocatalysis in flow
soluble hydrogenase, AtQR = quinuclidinone reductase).
for the production of chiral pharmaceutical molecules. The
manufacture of (R)-3-quinuclidol at a suitable purity has been a
key challenge for process chemists in recent years,[30–32] and this
is the first demonstration of a flow-based system to generate a
product with exceptionally high enantiopurity.
Conclusion
We have demonstrated an improved multi-phase flow reactor for
rapid heterogeneous biocatalysis with in-situ H
2
-driven cofactor
2
Continuous H -driven Biocatalytic Deuteration
recycling. Using only H gas as the reductant more closely
2
The biocatalytic flow system was next applied to the selective
resembles traditional chemo-catalysed routes, whilst overcoming
the high expense of using cofactors, and the waste generated by
traditional biocatalytic cofactor recycling methods. The main
challenge within this work was the stability of the SH activity within
the flow reactor, and so further development is required to
overcome this limitation. Through development of the reactor set-
up, overnight re-circulating reactions (>12 h) were improved to 2-
+
deuteration of NAD (Scheme 2A). Molecules bearing asymmetric
2
deuterium ( H) centers are desirable for medicinal and analytical
applications,[33,34] but are synthetically difficult to achieve. We
2
have identified H
2
-driven biocatalysis in heavy water, H
2
O, as a
new route to these targets.[35] The H
-driven system presented
2
here allows for biocatalytic deuteration without the need to co-
2
feed expensive pre-labelled compounds such as D-[1- H]-glucose
5
min tRes in a single-pass for >99% conversion. The flow reactor
2
2
and [ H
7
]-isopropanol, and without the need for H
2
gas. Instead,
O, >99%
was demonstrated as a simple method for the production of
important pharmaceutical fragments with exceptionally high ee. It
by simply exchanging the buffer from H
2
O to 2H
2
4
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ChemCatChem
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FULL PAPER
was also shown that simple modifications to the reaction set-up
allowed the flow-apparatus to be employed as a route to
isotopically labelled compounds.
[2]
M. Baumann, I. R. Baxendale, Beilstein J. Org. Chem. 2015, 11,
1194–1219.
[
[
[
[
3]
4]
5]
6]
A. R. Bogdan, A. W. Dombrowski, J. Med. Chem. 2019, 62, 6422–
6
468.
J. Britton, S. Majumdar, G. A. Weiss, Chem. Soc. Rev. 2018, 47,
891–5918.
Experimental Section
5
L. Tamborini, P. Fernandes, F. Paradisi, F. Molinari, Trends
Biotechnol. 2018, 36, 73–88.
General information: NAD+ and NADH were purchased from Prozomix.
Sodium pyruvate, ammonium chloride, 4’-chloroacetophenone and
Trizma-base were purchased from Sigma Aldrich. All reagents were used
M. B. Plutschack, B. Pieber, K. Gilmore, P. H. Seeberger, Chem.
Rev. 2017, 117, 11796–11893.
as received, without any additional purification. All solutions were prepared
2
with MilliQ water (Millipore, 18 MΩ cm) or H
2
O (99.98 %, Sigma Aldrich).
[7]
B. Gutmann, D. Cantillo, C. O. Kappe, Angew. Chemie - Int. Ed.
Deuterated buffer solutions were prepared by suspending Trizma-base in
2H
O and evaporating to dryness several times to exchange labile protons.
The solutions of [ H
addition of small quantities of 2HCl. UV-Vis spectra were obtained using
an Agilent Cary 60 spectrophotometer. 1H NMR spectra were obtained
2
015, 54, 6688–6728.
2
[
8]
B. Gutmann, C. O. Kappe, J. Flow Chem. 2017, 7, 65–71.
M. Irfan, T. N. Glasnov, C. O. Kappe, ChemSusChem 2011, 4, 300–
316.
2
2
5
]-Tris were then adjusted to the required p H with the
[9]
using either a Bruker Avance III HD nanobay (400 MHz) or a Bruker
[10]
P. J. Cossar, L. Hizartzidis, M. I. Simone, A. McCluskey, C. P.
Gordon, Org. Biomol. Chem. 2015, 13, 7119–7130.
V. Sans, L. Cronin, Chem. Soc. Rev. 2016, 45, 2032–2043.
D. C. Fabry, E. Sugiono, M. Rueping, React. Chem. Eng. 2016, 1,
129–133.
2
Avance III HD (500 MHz) instrument. For samples in H
2
O, 10% v/v H
2
O
was added for field locking purposes. GC-FID data was recorded using a
Thermo Scientific TRACE 1310 instrument, equipped with a CP-Chirasil-
Dex CB (Agilent), 25 m length, 0.25 mm diameter, 0.25 μm (film thickness),
fitted with a guard of 10 m deactivated fused silica of the same diameter.
Enzymes used: Soluble hydrogenase (SH) from Ralstonia eutropha,
alanine dehydrogenase (AlaDH) and quinuclidinone reductase from
Agrobacterium tumefaciens (AtQR) were prepared in-house.[35] Alcohol
dehydrogenase (ADH105, Johnson Matthey, Cambridge, UK) was used
as supplied.
[
[
11]
12]
[13]
G. A. Price, D. Mallik, M. G. Organ, J. Flow Chem. 2017, 7, 82–86.
P. Sagmeister, J. D. Williams, C. A. Hone, C. O. Kappe, React.
Chem. Eng. 2019, 4, 1571–1578.
[
14]
[
15]
R. Munirathinam, J. Huskens, W. Verboom, Adv. Synth. Catal.
2015, 357, 1093–1123.
General procedure for continuous flow reactions:
A detailed
preparation of the carbon nanotube column reactors has been described
previously.[26] The required mixture of enzymes was immobilised within the
carbon nanotube column reactor by adsorption at 4 °C. After 1 h, the
column was flushed with Tris-HCl buffer (50 mM, pH 8.0) to remove any
un-adsorbed enzymes. For the reactions to be carried out, the column
reactor (288 µL RV) was connected to the required pumps (Vapourtec V-
[16]
K. Masuda, T. Ichitsuka, N. Koumura, K. Sato, S. Kobayashi,
Tetrahedron 2018, 74, 1705–1730.
[
17]
M. Colella, C. Carlucci, R. Luisi, Top. Curr. Chem. 2018, 376, DOI
10.1007/s41061-018-0225-0.
[18]
[19]
R. A. Sheldon, S. van Pelt, Chem. Soc. Rev. 2013, 42, 6223–6235.
R. C. Rodrigues, C. Ortiz, Á. Berenguer-Murcia, R. Torres, R.
Fernández-Lafuente, Chem. Soc. Rev. 2013, 42, 6290–6307.
M. P. Thompson, I. Peñafiel, S. C. Cosgrove, N. J. Turner, Org.
Process Res. Dev. 2019, 23, 9–18.
3
1
peristaltic pumps, Syrris Asia syringe pump or Harvard Apparatus Model
1 syringe pump) using PTFE tubing (1/16” OD, 1/32” ID) and TefzelTM or
PEEK fittings (¼-28, 0.02” thru-hole, IDEX). In a typical reaction, a liquid
feed and a H gas feed were joined at a Y-piece (PEEK, ¼-28, 0.02” thru-
[
20]
2
hole, IDEX) before entering the column reactor. The flow rates of the
pumps were adjusted according to the desired tRes through the reactor
column, and with a gas:liquid flow rate ratio of approximately 3:1. All
reactions were carried out at room temperature. The reaction solution was
pumped through the reactor to waste for 2 x RV before samples were
collected for analysis.
Further details of the enzyme preparation and immobilisation, and
protocols for running continuous biocatalytic reactions are provided in the
SI, together with 1H NMR, chiral GC and UV-Vis spectroscopic
characterisation of the products.
[21]
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Entry for the Table of Contents
Biocatalytic hydrogenations give high conversion and high enantiomeric excess under mild conditions, in a single pass through an
enzyme-modified carbon nanotube lined column. This can also be applied to selective reductive deuterium incorporation.
Institute and/or researcher Twitter usernames: @vincentgroupox, @HydRegen_Oxford
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