Accelerating Biphasic Biocatalysis through New Process Windows

Article abstract of DOI:10.1002/ange.202005183

Process intensification through continuous flow reactions has increased the production rates of fine chemicals and pharmaceuticals. Catalytic reactions are accelerated through an unconventional and unprecedented use of a high-performance liquid/liquid counter current chromatography system. Product generation is significantly faster than in traditional batch reactors or in segmented flow systems, which is exemplified through stereoselective phase-transfer catalyzed reactions. This methodology also enables the intensification of biocatalysis as demonstrated in high yield esterifications and in the sesquiterpene cyclase-catalyzed synthesis of sesquiterpenes from farnesyl diphosphate as high-value natural products with applications in medicine, agriculture and the fragrance industry. Product release in sesquiterpene synthases is rate limiting due to the hydrophobic nature of sesquiterpenes, but a biphasic system exposed to centrifugal forces allows for highly efficient reactions.

Full text of DOI:10.1002/ange.202005183

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Titel: Accelerating biphasic biocatalysis through new process windows
Autoren: Florence Huynh, Matthew Tailby, Aled Finniear, Kevin
Stephens, Rudolf K Allemann, and Thomas Wirth
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Zitierweise: Angew. Chem. Int. Ed. 10.1002/anie.202005183
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10.1002/ange.202005183
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COMMUNICATION
Accelerating biphasic biocatalysis through new process windows
Florence Huynh,[a] Matthew Tailby,[a] Aled Finniear,[b] Kevin Stephens,[b] Rudolf K. Allemann*[a] and
Thomas Wirth*[a]
Abstract: Process intensification through continuous flow reactions
has increased the production rates of fine chemicals and
pharmaceuticals. Catalytic reactions are accelerated through an
Recent developments have made robust and reliable CCC
instruments commercially available.[7] This technique relies on the
partition of solutes between two immiscible liquid phases therefore
avoiding the solid phases used in many other chromatographic
procedures. One liquid phase is held stationary in a coiled tube
through centrifugal forces by spinning the whole coil. The second
(mobile) liquid phase, immiscible with the stationary liquid phase, is
pumped through the system.[8] The high-performance counter current
chromatography (HPCCC) system uses a polytetrafluoroethylene
(PTFE) tube wrapped in a coil onto a drum (bobbin) which rotates in
a planetary motion (Figure 1). The bobbin revolves around its central
axis while simultaneously rotating around its own axis at the same
velocity to create an effect similar to wave mixing with more than two
million partitioning steps per hour.[9] As this dramatically increases the
liquid/liquid interfacial area, we hypothesized that it can provide an
ideal platform to improve synthetic processes and opening up
alternative and new process windows that rely on biphasic mixing.
unconventional and unprecedented use of
a high-performance
liquid/liquid counter current chromatography system. Product
generation is significantly faster than in traditional batch reactors or in
segmented flow systems, which is exemplified through
stereoselective phase-transfer catalyzed reactions. This methodology
also enables the intensification of biocatalysis as demonstrated in
high yield esterifications and in the sesquiterpene cyclase-catalyzed
synthesis of sesquiterpenes from farnesyl diphosphate as high-value
natural products with applications in medicine, agriculture and the
fragrance industry. Product release in sesquiterpene synthases is rate
limiting due to the hydrophobic nature of sesquiterpenes, but a
biphasic system exposed to centrifugal forces allows for highly
efficient reactions.
Flow chemistry is changing the way chemical reactions are performed.
The development of catalytic processes in flow chemistry has already
led to major advances in the synthesis of fine chemicals and
pharmaceuticals. The involvement of heterogeneous catalysts in flow
systems has a long history and different catalyst-containing reactors
have been developed for these reactions.[1] Homogenously catalyzed
reactions have high value also from the industrial perspective,[2] but
are more difficult to control in flow reactions as catalyst recycling
methods usually have to be implemented to obtain sustainable
protocols. Different approaches have been reported for the
immobilization (heterogenization) of homogeneous catalysts but often
the catalytic reactivity and selectivity are compromised.
Introduced by V. Hessel,[3] the concept of ‘novel process windows’
describes flow procedures operating under unusual reaction
conditions. They can overcome traditional thermodynamic or kinetic
limitations and, therefore, result in intensified processes or even novel
reaction outcomes.
Efficient mixing can sometimes be challenging to achieve, especially
in an industrial setting. Mixing can influence the outcome of chemical
reactions as product selectivities can be altered through competitive
parallel or competitive consecutive reactions.[4] Many such reactions
have been described in the literature; the trapping of reactive and
unstable intermediates though rapid mixing is one prominent
example.[5]
Figure 1. High performance counter current chromatography (HPCCC) device.
(a) Schematic view showing only one of the two bobbins for clarity. (b) Settling
and mixing zones in the HPCCC column generated by variable centrifugal
forces induced by the planetary motion of the bobbin. The mixing zone is
situated toward the centre of the axis of revolution coinciding with a low
acceleration field and the settling zone coinciding with the high acceleration field
away from the centre of revolution.
Liquid/liquid counter current chromatography (CCC) has been
developed for the separation of mixtures of natural compounds or for
the purification of pharmaceuticals on analytical to industrial scales.[6]
[a]
[b]
F. Huynh, M. Tailby, Prof. Dr. R. K. Allemann, Prof. Dr. T. Wirth
School of Chemistry, Cardiff University
Main Building, Park Place, Cardiff, CF10 3AT (UK)
E-mail: allemannrk@cardiff.ac.uk; wirth@cardiff.ac.uk
Dr. A. Finniear, K. Stephens, Bioextractions (Wales) Ltd.,
Trafarnaubach, Tredegar (UK)
The use of small-scale capillaries to improve mixing of biphasic
systems through segmented flow as opposed to rapid stirring in a flask
has already been characterized by mass transfer coefficients and
modelled by engineers.[10] Even the geometry of the capillary can
improve mixing by up to 20% through Dean forces.[11] Modelling of
Supporting information for this article is given via a link at the end of
the document.
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COMMUNICATION
HPCCC systems however, is more complex and different models
have been proposed for their theoretical investigation.[12]
conditions provided product 2 in 26% yield and with 63% e.e. with a
residence time of 23.5 minutes. The same solvents were used for
experiments in the HPCCC where 54% yield of 2 (65% e.e.) was
obtained in a reaction with 12 minutes residence time. All reactions
were performed at room temperature and the obtained
enantioselectivities were similar. With catalyst 3b, which shows
enhanced solubility (0.003 M in toluene) and which had been used
previously in epoxidation reactions[17] but not in the stereoselective
alkylation, selectivity was greatly improved (Figure 2). Further
optimization was performed (see supporting information) with regards
to flow rate / retention time in the machine. The optimal reaction time
of 10.7 minutes was then applied to the batch and segmented flow
protocols showing unambiguously the large improvement obtained in
HPCCC with 73% yield and 87% e.e. of 2.
Phase-transfer catalysis has become a well-established synthetic
technique since its discovery in the 1960s.[13] With two reactants being
present in two separate immiscible phases, the role of a phase-
transfer catalyst (PTC) is to enable the movement of one reactant
from one phase into the other to increase the reaction rate
substantially. For stereoselective reactions, chiral PTCs can be used
to generate products in high selectivity.[ 14 ] Enhanced mixing in
microreactors has already been exploited for phase-transfer
catalyzed reactions.[15] Here, we report the first stereoselective phase-
transfer catalyzed reaction in a flow system using intensified mixing
with the HPCCC device. The stereoselective alkylation of N-
(diphenylmethylene)glycine tert-butyl ester 1 uses 50% aqueous KOH
as base, 5 equivalents of benzyl bromide (BnBr) and a chiral PTC.
This is a well-explored reaction leading to product 2 in good yields and
selectivities where cinchona-derived catalysts have been successfully
used.[16] Products of type 2 can be hydrolyzed to optically active amino
acid derivatives as useful building blocks for further synthesis.
Different alkyl halides were investigated in the reaction using the
HPCCC. With residence times between 10 and 16 minutes, products
4 were all obtained with high enantioselectivities characteristic for
catalyst 3b (Figure 3). Allylic bromides and propargylic bromides were
suitable electrophiles too (4g–4i), while alkyl bromides or benzyl
chlorides were unreactive under the conditions used. Even alkyl
iodides were unreactive, although these had been reported previously
to be used successfully in long (30 hours) batch alkylations.[ 18 ]
Reactions with extremely long reaction times cannot be performed on
the HPCCC. The biphasic stereoselective epoxidation of chalcone
with sodium hypochlorite, for which catalyst 3b was originally
prepared, takes 48 hours in batch and this reaction could not be
performed successfully by HPCCC.[17]
aq. KOH (50%)
BnBr (5 eq.)
Ph
N
CO2tBu
Ph
N
CO2tBu
catalyst 3 (10 mol%)
Ph
Ph Bn
2
1
HO
BnO
N+
Bn
N+
Br–
Cl–
N
N
3a
3b
aq. KOH (50%), toluene
RBr (5 eq.)
catalyst 3b (10 mol%)
Ph
N
CO2tBu
Ph
Ph
N
CO2tBu
a
b
c
HPCCC
batch
flow
Ph
Ph
R
HPCCC
1
4
CHCl3 / toluene 7:3
50% aq. KOH
reaction time: 7 h
toluene
50% aq. KOH
reaction time: 23.5 min
toluene
50% aq. KOH
reaction time: 12 min
Ph
N
CO2tBu
Ph
N
CO2tBu
N
CO2tBu
Ph
Ph
Ph
cat. 3a:
cat. 3b:
2, 62% yield, 66% e.e. 2, 26% yield, 63% e.e.
2, 62% yield, 87% e.e. 2, 50% yield, 89% e.e.
2, 54% yield, 65% e.e.
2, 57% yield, 86% e.e.
CF3
4a, 67% yield, 87% e.e.
CO2Me
4b, 57% yield, 87% e.e.
NO2
4c, 60% yield, 81% e.e.
reaction time: 10.7 min reaction time: 10.7 min
reaction time: 10.7 min
2, 73% yield, 87% e.e.
cat. 3b: 2, 10% yield, 86% e.e. 2, 26% yield, 86% e.e.
Ph
N
CO2tBu
Ph
N
CO2tBu
Ph
N
CO2tBu
Scheme 1. Stereoselective phase-transfer catalysis. (a) Batch reaction. (b)
Segmented flow reaction. (c) HPCCC reaction.
Ph
Ph
Ph
Ph
4d, 75% yield, 81% e.e.
I
4e, 60% yield, 87% e.e.
4f, 53% yield, 81% e.e.
Initial investigations of the batch reactions showed a low solubility of
catalyst 3a. Therefore, the reported solvent system (toluene:chloro-
form 7:3) was changed to dichloromethane where the solubility of 3a
is 0.005 M. In a biphasic reaction in segmented flow, comparable
yields and selectivities were obtained with an optimized internal
diameter (0.5 mm), flow rate (0.32 mL/min) and residence time (21
min). Unfortunately, this solvent system cannot be used in the HPCCC
as the interfacial tension between the two phases is too small and an
emulsion is formed. Toluene would be a suitable organic solvent in
the HPCCC due to the large density difference to 50% aq. KOH, but
the catalyst is not soluble in either of these solvents. Although the
catalyst is soluble in 25% aq. KOH, only trace amounts of product
were formed. Preforming the enolate of 1 and exchanging it with the
chloride anion of 3a allowed the formation of a homogenous solution
of the catalyst in toluene and the reaction under segmented flow
Ph
N
CO2tBu
Ph
N
CO2tBu
Ph
N
CO2tBu
Ph
Ph
Ph
4g, 61% yield, 80% e.e.
4h, 40% yield, 81% e.e.
4i, 25% yield, 81% e.e.
Scheme 2. Different electrophiles in the stereoselective phase-transfer
catalyzed alkylation.
In a previous publication, the use of counter current extraction devices
in biocatalysis was described. However, only little details were given
in that seminal publication[19] and later reports describe only the use
of the centrifugal partition chromatography equipment as an
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intensified reactor for biocatalytic reactions.[ 20 ] We investigated
HPCCC as an alternative method to overcome the limitations of
traditional batch and flow approaches in biocatalysis.
biphasic mixture to release the product into an organic phase, a
method which has been used with success also on other enzymes
such as lipases.[28] However, this has proven to be inefficient for
sesquiterpene synthases due to a low mass transfer rate and a
deactivation of the enzyme through long exposure to organic solvents.
Typical yields range from 10 to 30% and reaction times are often long
(1–2 days). For optimum yields the substrate needs to be added in
stages and reaction products extracted several times with organic
solvents, which often leads to the formation of emulsions requiring
centrifugation for separation. As an alternative, we have reported the
use of sesquiterpene synthases in segmented flow systems to
accelerate mixing and to shorten reaction times.[ 29 ] Optimization
through design of experiments improved the yields and shortened the
reactions times for the conversion of farnesyl diphosphate 10 to (+)-
aristolochene (11) and amorpha-4,11-diene (13) by aristolochene and
amorphadiene synthase in 96% and 69% yields respectively in 90
minutes.[30] Despite improved yields and reaction rates, segmented
flow systems can still result in emulsions at the reactor outlet with
occasional blockages during extended reactions due to enzyme
precipitation through contact with pentane.
In initial experiments we used Cal B lipase for the transesterification
of octanol with vinyl acetate. The organic phase (heptane) contained
octanol 5 and vinyl acetate 6 while the aqueous buffer contained the
Cal B lipase. Both solutions were pumped through the HPCCC.
Rotation speed in the HPCCC, residence time and temperature were
varied in a face-centered design of experiments (DoE) approach (see
supporting information), taking into account that only a smaller
amount of the stationary phase is retained under these conditions. A
three-fold excess of vinyl acetate with an enzyme concentration of 1
mg•mL–1 and operating the HPCCC at maximum rotation speed (1600
rpm) led to an almost quantitative isolated yield (97%) of octyl acetate
7 with a 6 minutes reaction time. Compound 7 is an industrially
important solvent but is also found in citrus fruits and used as the basis
for artificial flavors. If the same reaction is carried out in a stirred flask,
only 40% yield of 7 is obtained after 4.5 hours reaction time. The
reaction of rac-pentan-2-ol 8 with vinyl acetate and Cal B lipase led to
a
racemic resolution of 8 and the quantitative formation of
enantiomerically pure (R)-9. While the reported batch reaction takes
4 hours to complete,[21] the reaction time in the HPCCC is only 6
minutes.
Here we examined the conversion of 10 to (+)-aristolochene by aristo-
lochene synthase from Penicillium roqueforti (AS). Purified enzyme
and 10 in a buffer solution were loaded on the HPCCC analytical
column (22 mL volume) to form a stationary phase (see supporting
information). Buffer, substrate and enzyme concentration were
identical to those previously used for batch and segmented flow
experiments. To assess the efficiency of HPCCC, it was first
determined how long the mobile phase needed to be flowed through
the system to reach a plateau in product extraction. Surprisingly,
>95% yield (measured by GC) was reached in less than one column
volume (CV) at a flow rate of the mobile phase of 0.5 mL•min–1. The
first phase shown in blue in Figure 5b is the equilibration of the
biphasic system (definition of equilibration and elution step in the
HPCCC are detailed in the supporting information). To investigate
further the potential of HPCCC for synthesis, the concentration of
substrate 10 was doubled (0.7 mM) to increase the scale of the
reaction. This had no impact on the turnover as a similar yield was
obtained after 0.5 CV (Figure S4).
Cal B lipase
20 mM phosphate
buffer, pH 7.2
OH
5
OAc
OAc
6
+
HPCCC
7, 97% yield
Cal B lipase
20 mM phosphate
buffer, pH 7.2
OH
OAc
+ (S)-8
OAc
+
rac-8
6
(R)-9, 49% yield
>99% e.e.
HPCCC
Scheme 3. Transesterifications with Cal B lipase.
Sesquiterpenes belong to a class of natural products with more than
300 known hydrocarbon backbones. Many sesquiterpene derivatives
exhibit important bioactivities with medicinal or agricultural
applications.[ 22 ] In nature, these compounds are synthesized by
sesquiterpene synthases in a single step from farnesyl diphosphate
(10) in exquisitely regioselective and stereospecific reactions (Figure
5a).[23] Despite their high value, most sesquiterpenes are not available
in large quantities as they have to be extracted from plants. They are
not easily available through total synthesis due to challenges
associated with stereo- and regioselectivity and they are often
unstable to heat and acidic conditions. Although recent synthetic work
has shown promising progress,[24] nature is much more efficient in
producing these complex compounds.[ 25 ] Therefore, recombinant
sesquiterpene synthases expressed in E. coli have been used to
synthesize natural sesquiterpenes and to expand the terpenome in
the search for novel compounds. Unnatural analogues of farnesyl
diphosphate have been successfully converted to new structures
including oxygenated products such as aldehydes and cyclic
ethers.[ 26 ] The bottleneck of this approach is the typically high
hydrophobicity of the sesquiterpene products, which can lead to slow
reactions. Pre-steady state kinetic measurements have shown that
product release is normally the rate-limiting step of the overall
reaction.[27] An initial solution was the creation of a rapidly stirred
The force exerted on the retained phase is closely correlated to its
density, the revolving speed of the coil and the flow rate of the mobile
phase. When the flow rate was increased from 0.5 mL•min–1 to 2
mL•min–1 (reducing the extraction time to a quarter), the increased
flow rate affected the retaining force exerted on the aqueous phase
leading to a loss of the stationary phase. The percentage retention of
the stationary phase relative to the total column volume is defined as
Sf. Glycerol is usually required to maintain the stability of
sesquiterpene synthases in flow and batch, but the additional density
it provides reduced Sf (Figs. 5c–d). However, the reaction time when
using HPCCC is so short that glycerol can be omitted. With the
modified buffer conditions and a rotation speed of 1600 rpm,
approximately half of the column volume (Sf = 50%) can be retained
as stationary phase at a pentane flow rate of 2 mL•min–1. This
increased flow rate did not negatively impact on the yield (Table S7)
and reduced the reaction time from 44 to 11 minutes. Thus, HPCCC
was clearly more efficient than segmented flow and is characterized
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COMMUNICATION
a
OPP
H
H
AS
GDS
ADS
10
10
10
10, farnesyl diphosphate
11, (+)-aristolochene
12, (S)-germacrene D
13, amorpha-4,11-diene
b
c
d
Figure 2. Sesquiterpene cyclases in HPCCC. (a) Conversion of farnesyl diphosphate 10 to 11, 12 and 13 catalyzed by aristolochene (AS), (S)-germacrene D (GDS)
and amorphadiene (ADS) synthases. (b) Time course for the AS-catalyzed conversion of 10 to 11 in the HPCCC system (6 μM AS, 0.35 mM 1, 0.5 mL•min–1
pentane flow rate), determined by GC-FID. [a] Yield of 11 in total eluted volume. (c) Characterization of the relationship between the pentane flow rate and glycerol
content of the aqueous phase on the stationary phase retention Sf. The surface highlights that Sf is severely reduced with glycerol in the buffer and a high flow rate.
(d) Characterization of the relationship between the pentane flow rate and rotation speed of the bobbin on the stationary phase retention Sf. Surface highlights that
Sf is severely reduced with low rotation speed and high flow rate. (Surface rendered using Matlab 2018b). Sf: Percentage retention of the stationary phase relative
to total column volume.
by a ~10-fold reduced reaction time. We then applied these optimized
Figure 3. Comparison of batch, segmented flow and HPCCC methods for
conversions of FDP (10) with aristolochene synthase (AS), amorphadiene
synthase (ADS) and (S)-germacrene D synthase (GDS) and for 12-OH-FDP
with ADS. Yields were determined by GC and calculated by using a calibration
HPCCC conditions to different sesquiterpene cyclases using the
natural substrate 10 as well as modified analogues of 10 such as 12-
hydroxy farnesyl diphosphate (12-OH FDP). Vastly improved yields of
up to 99% were observed compared to batch synthesis, where yields
were typically between 20 and 40% (Figure 6 and supporting
information). HPCCC has been previously reported to scale well when
used for purification[31] and the optimized conditions for synthesis were
investigated with alarger HPCCC column (135 mL). A similar
extraction profile to the smaller column was obtained (Figure S3) with
excellent isolated yields of 70–94% (30–35 mg) (Figure 6),[32] in good
agreement with those measured by GC for the smaller HPCCC
column.
curve with a-humulene as standard. Isolated yields of the products have been
obtained using the preparative scale (135 mL) HPCCC column with optimized
reaction conditions (pentane flow rate: 5 mL•min–1).
In summary, we have demonstrated here for the first time that HPCCC
provides a unique and general platform to perform biphasic reactions,
enabling extremely rapid mass-transfer between two immiscible
phases. This method was applied to phase-transfer catalyzed
chemical alkylations and biocatalytic transformations. The reactions
with Cal B lipase and with sesquiterpene synthases produce valuable
products with high yields and with extremely short reaction times. The
sesquiterpene catalyzed reactions were approximately 70 times faster
in HPCC than in batch. The short reaction times avoid enzyme
denaturation alleviating emulsion formation seen in batch and
segmented flow systems. This method is readily scaled up with larger
HPCCC columns and will find utility with many other reactions where
products and / or substrates are poorly water soluble.
batch
flow
HPCCC
Experimental Section
Enzyme overexpression and purification: Recombinant aristolochene
synthase from Penicillium roqueforti, amorphadiene synthase from
Artemisia annua and (S)-germacrene
canadensis were prepared as previously described.
concentrations were measured by Bradford assay and enzymes stored at
–20 °C.[34]
D
synthase from Solidago
H
H
H
H
[26c, 33 ]
Protein
CHO
14, 70% yield
11, 94% yield
13, 84% yield
12, 92% yield
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Synthesis of 1: FDP (1) was synthesized from commercial (2E,6E)-
farnesol using the method described by Davisson.[35] 12-Hydroxy FDP (12-
OH-FDP) was synthesized from commercial (2E,6E)-farnesol using the
method of Demiray et al.[26c]
Acknowledgements
We thank BioExtractions (Wales), the KESS 2 program (M. T.)
and the School of Chemistry, Cardiff University (PhD studentship
to F. H.), for financial support. Knowledge Economy Skills
Scholarships (KESS) is a pan-Wales higher level skills initiative
led by Bangor University on behalf of the HE sector in Wales. It is
part funded by the Welsh Government’s European Social Fund
(ESF) convergence programme for West Wales and the Valleys.
We are grateful to Dr. R. L. Jenkins and T. Williams for assistance
with mass spectrometry and gas chromatography. We thank T.
Drevon for rendering Figs. 1 and 5c–d.
General procedure for biocatalysis using HPCCC: All experiments were
performed on a Dynamic Extractions SpectrumTM instrument (Slough, UK)
fitted with an analytical scale column with a volume of 22 mL (0.8 mm I.D.
PTFE tubing) and a revolution radius of 85 mm. Semi-preparative scale
experiments used a column with a volume of 135 mL (1.6 mm I.D. of 1.6
mm PTFE tubing) and a revolution radius of 85 mm. Aqueous and organic
phases circulated with two HPLC pumps (ECOM ECP 2010) at 28 ºC. The
column was initially filled with pentane (HPLC grade, Sigma-Aldrich) at 6
mL•min–1 from tail periphery to head-centre. The column was then loaded
through a loop with 10 mL AS incubation buffer (20 mM Tris-base, 3 mM
MgCl2, pH 7.5) containing 6 µM AS and 0.35 mM FDP at a flow rate of
2 mL•min–1 resulting in 10 mL of pentane being displaced from the column.
The instrument was rotated at 1600 rpm and pentane was pumped from
tail periphery to head-centre at a flow rate of 2 mL•min–1. The pentane was
collected in 5 mL fractions and analysed by GC. The yield was calculated
by comparing peak areas of the product to a calibration curve using a-
humulene.
Keywords: Biocatalysis • Biphasic reaction • HPCCC • Phase-
transfer catalysis • Terpenoids
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Entry for the Table of Contents
COMMUNICATION
A new application of high-performance
liquid/liquid counter current chromato-
graphy system demonstrated high
reaction rates and excellent yield
improvements for biphasic reactions,
exemplified with steroselective phase-
transfer catalysed reactions and with
the biocatalytic synthesis of sesqui-
terpenes. Catalysis in the HPCCC
system was up to 70 times faster than
in batch reactors and up to 10 times
faster than in segmented flow systems
with nearly quantitative yields.
F. Huynh, M. Tailby, A. Finniear, K.
Stephens, R. K. Allemann,* T. Wirth*
water
pentane
H
H
O
P
O
Page No. – Page No.
P
O
O
O
O
O
HPCCC
current counter extraction
70 x faster than
Accelerating biphasic biocatalysis
through new process windows
batch reactor
Ph
N
CO2tBu
BnBr
Ph
N
CO2tBu
cat*
Ph
Ph Bn
+
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