An alternative approach towards poly-ε-caprolactone through a chemoenzymatic synthesis: Combined hydrogenation, bio-oxidations and polymerization without the isolation of intermediates

Article abstract of DOI:10.1039/c6gc02529c

A novel synthetic route towards the polymer poly-ε-caprolactone based on a chemoenzymatic reaction sequence was developed. Initial hydrogenation of phenol to cyclohexanol gave a crude product, which was directly used without work-up for a subsequent biocatalytic double oxidation towards ε-caprolactone by means of an alcohol dehydrogenase and a monooxygenase. In order to overcome product inhibition effects, an in situ-product removal strategy via extraction of ε-caprolactone from an aqueous reaction medium with an organic solvent in the presence of a permeable polydimethylsiloxane membrane was applied. Furthermore, this in situ-product removal was combined with lipase-catalyzed polymerization in the organic phase at 25 °C. The obtained crude product contained a polymer fraction with a degree of polymerization comparable to commercial poly-ε-caprolactone.

Full text of DOI:10.1039/c6gc02529c

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An alternative approach towards poly-ε-
caprolactone through a chemoenzymatic
synthesis: combined hydrogenation, bio-
oxidations and polymerization without the
isolation of intermediates†
Cite this: DOI: 10.1039/c6gc02529c
Severin Wedde,a Philipp Rommelmann,a Christian Scherkus,b Sandy Schmidt,c
Uwe T. Bornscheuer,c Andreas Liese*b and Harald Gröger*a
A novel synthetic route towards the polymer poly-ε-caprolactone based on a chemoenzymatic reaction
sequence was developed. Initial hydrogenation of phenol to cyclohexanol gave a crude product, which
was directly used without work-up for a subsequent biocatalytic double oxidation towards ε-caprolactone
by means of an alcohol dehydrogenase and a monooxygenase. In order to overcome product inhibition
effects, an in situ-product removal strategy via extraction of ε-caprolactone from an aqueous reaction
medium with an organic solvent in the presence of a permeable polydimethylsiloxane membrane was
applied. Furthermore, this in situ-product removal was combined with lipase-catalyzed polymerization in
the organic phase at 25 °C. The obtained crude product contained a polymer fraction with a degree of
polymerization comparable to commercial poly-ε-caprolactone.
Received 9th September 2016,
Accepted 13th December 2016
DOI: 10.1039/c6gc02529c
www.rsc.org/greenchem
design for a sustainable production of today’s existing petro-
chemical-based polymers by combining chemo- and biocataly-
Introduction
As a key challenge for the next few decades in biocatalysis one sis would be attractive.4 Towards this end, many efficient
can regard the integration of biotransformations within chemi- chemo- and bio-catalytic technologies, which are already avail-
cal multi-step reaction sequences and the application of such able, can serve as modules for such new chemoenzymatic reac-
chemoenzymatic processes for the production of bulk chemi- tion sequences.5
cals. Although biocatalysis has already emerged as an impor-
In the following, we report an alternative synthetic
tant synthetic tool in the production of chiral fine chemicals approach towards the petrochemical polymer poly-ε-capro-
and pharmaceuticals, its application in the field of bulk lactone (PCL) starting from phenol and based on the combi-
chemicals is still rare.1 The integration of biocatalytic key steps nation of four synthetic key steps comprising metal-catalysed
in chemical multi-step sequences and the design of novel ret- as well as enzymatic reactions. It is noteworthy that besides
rosynthetic pathways towards industrial chemicals might also from crude oil3 phenol is accessible from lignocellulose via
contribute to switch the chemical raw material supply from a pyrolysis6 or glucose via fermentation,7 thus enabling in prin-
petrochemical to a biorenewable basis.2 Since the largest pro- ciple the synthesis of PCL also from a biorenewable starting
duction volumes of industrial chemicals typically relate to material.
applications in the polymer sector,3 such an alternative route
In detail, the process concept (which is shown in Scheme 1)
is based on an initial access to phenol (via the Hock process
starting from benzene or by conversion of a biorenewable feed-
stock),3,6,7 followed by a metal-catalysed hydrogenation to
cyclohexanol and subsequent bi-enzymatic transformation
into ε-caprolactone (ε-CL, 4). In this biotransformation, first
cyclohexanol is oxidized by means of a dehydrogenase to cyclo-
hexanone under consumption of NADP+, thus forming
NADPH. In the second enzymatic step, namely oxidation of
cyclohexanone to ε-CL with a monooxygenase, NADPH is con-
verted back into NADP+. Thus, NADP+ is in situ regenerated
aChair of Organic Chemistry I, Faculty of Chemistry, Bielefeld University,
Universitätsstrasse 25, 33615 Bielefeld, Germany.
E-mail: harald.groeger@uni-bielefeld.de
bInstitute of Technical Biocatalysis, Hamburg University of Technology, Denickestr.
15, 21073 Hamburg, Germany. E-mail: liese@tuhh.de
cInstitute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis, University of
Greifswald, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6gc02529c
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Table 1 Phenol hydrogenation with the heterogeneous catalyst Rh(0)-
SBA-15
Entry
Rh cat. [mol%]
t [h]
1
c [%]
2
c [%]
3
c [%]
1a
2a
3b
4b
1.9
1.0
1.0
0.5
24
20
24
24
1
1
1
99
99
98
16
0
0
1
57
27
a 0.5 M substrate concentration. b 1.0 M substrate concentration. c The
relative amount of each of the compounds 1, 2 and 3, respectively,
after the reaction is over.
without the isolation of intermediate 2 (according to the reac-
tions shown in Scheme 1). After conducting the hydrogenation
and removal of the hydrogen atmosphere as well as sub-
sequent separation of the catalyst by filtration, the aqueous
cyclohexanol solution with traces of phenol and cyclohexanone
was diluted (for reasons of enzyme inactivation and inhibition
at a higher substrate concentration)9a,b and then used directly
for oxidation to ε-CL (4) by means of a cyclohexanone mono-
oxygenase from Acinetobacter sp. NCIMB 9871 (CHMO) and an
alcohol dehydrogenase from Lactobacillus kefir (Lk-ADH) as
isolated enzymes just requiring O2 as the reagent and fully
avoiding a co-substrate (Fig. 1). It is noteworthy that the results
for ε-CL from this integrated process without cyclohexanol
Scheme 1 Concept for a sustainable synthesis route towards the pro-
duction of PCL (5) from phenol (being accessible from biorenewable
sources) by combination of metal-catalyzed hydrogenation and multi-
enzymatic steps.
and can be used in catalytic amounts. The final step then
consists of an in situ removal of ε-CL without work-up and
direct polymerization of ε-CL towards PCL. Since for the initial
access to phenol established approaches are available,3,6,7 our
major interest was to disclose a proof of concept for a combi-
nation of a metal-catalysed hydrogenation and subsequent
enzymatic steps without the need for isolation and purification
of any of the intermediates.
Results and discussion
To start with the initial hydrogenation step, such reactions
with aromatic compounds are extensively described in the lit-
erature.8 Heterogeneous catalysts offer the advantage of excel-
lent product removal and high recycling potential. The catalyst
Rh(0)-SBA-15,8f consisting of rhodium nanoparticles entrapped
in mesoporous silica gel, meets these requirements in general
and was used in our study. A screening of the reaction con-
ditions led to an optimized reduction process of phenol (1)
towards cyclohexanol (2; Table 1). Our best result was obtained
with a 1 M aqueous phenol solution, using 1 mol% active
Rh(0) and 1 bar hydrogen at 75 °C for 24 h, which yielded a
conversion of 99% with a selectivity of 99% for cyclohexanol
(Table 1, entry 3).
Fig. 1 Biocatalytic oxidation of 2 prepared in situ via hydrogenation of
phenol (blue-colored symbols: results from the reaction with commer-
cially available cyclohexanol; orange-colored symbols: results from the
The optimized hydrogenation process was then combined
with our developed enzymatic “double oxidation” method9a,b reaction with cyclohexanol from the hydrogenation process).
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isolation are comparable to the one for the biotransformation
with commercially available, purified cyclohexanol (1) as the
substrate (Fig. 1). Almost 90% reaction yield for the desired
ε-CL (4) could be achieved in both cases after 24 h reaction
time. Thus, a residual amount of phenol in the reaction
mixture does not influence the enzyme activities negatively.
These results show that the “back integration” of ε-CL
synthesis with preceded phenol hydrogenation has no negative
impact on the enzymatic oxidation cascade.
Next, we focused on the biocatalytic double oxidation
step9–11 as in previous work, it turned out that the process
efficiency is hampered by inactivation as well as inhibition
especially of the CHMO from Acinetobacter sp. by substrate 2,
intermediate 3 and product 4.9a,b Substrate inhibition could be
addressed by a fed-batch strategy, and product inhibition was
taken into consideration by an in situ-product removal
approach through oligomerization of ε-CL (4) via lipase A from
Candida antarctica in the aqueous reaction medium to its oli-
gomers.10a An initial study of water–organic two phase systems
for in situ-product removal from the aqueous phase revealed
that hydrophobic solvents with high log P-values were suitable
as co-solvents when being used in a low amount,9c but more
detailed studies showed severe negative effects when increas-
ing the organic solvent amount of such two-phase systems (for
details, see the ESI†). The observed decrease in conversion
might be attributable to a deactivating effect of the phase Fig. 2 (A) Photograph of a PDMS thimble. (B) Selectivity of the PDMS-
membrane. (C) Whole-cell catalytic biotransformation of cyclohexanol
to ε-CL in a pure aqueous buffer (blue) and a PDMS-thimble system
(two-compartment one-pot system) with selective component
exchange (orange). The compartment inside the PDMS-thimble contains
boundary on the enzymes.
In order to overcome these limitations when using organic
co-solvents, we focused on a new access towards the bulk
chemical ε-CL and its polymer product PCL through the com-
the aqueous solution for the biotransformation. This compartment is
bination of the concepts “in situ-product removal” and “mem- selectively separated by the PDMS-membrane from methylcyclohexane
(outside the thimble) for in situ extraction.
brane-based solvent compartmentation”. The organic solvents
were chosen based on the criterion of environmental sustain-
ability and also based on their classification of being at least
“acceptable”.12 For in situ extraction we used the following sol- able only to methylcyclohexane. Thus, methylcyclohexane was
vents as such with differing log P values: methyl-tert-butylether selected as the solvent of choice in order to protect the
(MTBE; log P 0.94), cyclohexane (log P 3.44) and methyl- enzymes from the deactivating effect of the extracting solvent
cyclohexane (log P 3.88).12 To avoid the contact of the aqueous (Fig. 2B).
with the organic solvent during in situ extraction when con-
Having a promising membrane-based extraction system
ducting the biocatalytic double oxidation, we studied the without a direct contact of the aqueous and organic phase in
usage of a polydimethylsiloxane (PDMS)-thimble13 as a selec- hand, we next carried out biotransformations with recombi-
tively permeable membrane (Scheme 1 and Fig. 2A). First we nant whole-cells at 40 and 100 mM substrate concentrations.
investigated the diffusion of cyclohexanol (2), cyclohexanone As at a substrate loading of 40 mM of cyclohexanol in a pure
(3) and ε-CL (4) from the aqueous reaction compartment aqueous system the effect of the substrate and product inhi-
(inside the thimble) through the thimble membrane into the bition as well as enzyme inactivation was found not to be criti-
organic compartment (outside of the thimble) containing the cal,9 in accordance with this previous result9 nearly quantitat-
extracting solvent. Independent of the organic solvent used, ive conversion was observed in the case of 40 mM of 2
the PDMS membrane of 200–250 μm thickness is permeable (Fig. 2C). In contrast, at an increased substrate concentration
for all reaction components 2–4 (Fig. 2B).
of 100 mM of 2 these negative effects become evident and con-
Although ideally it would be preferred to use a membrane, version dramatically drops to 50%. When using the PDMS-
which is exclusively permeable for ε-CL (4) alone, diffusion of thimble system with separated aqueous and organic phases
substrate 2 and intermediate 3 is not critical as the membrane under the conditions of in situ-product removal, at 40 mM sub-
is permeable in both directions, and the substrate con- strate loading a similar conversion of 97% was found, thus
centration can be kept low by applying a fed-batch mode. indicating a proof of concept for the suitability of this method-
However, analysis of the aqueous phase revealed that among ology. Furthermore, the advantage of the thimble system
the organic solvents applied the PDMS-membrane is imperme- emerges at 100 mM substrate loading by an increase of conver-
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sion from 50% to 99% (Fig. 2C), thus indicating that product thesis could be applied (dependent on the demand) for the
inhibition and deactivation as well as the negative impact of production of ε-CL (4, used as a monomer but also needed as
the organic solvent on the biocatalyst can be avoided via com- a solvent) or alternatively directly the polymer PCL (5). The
partmentation of the aqueous phase (for the biotransform- lipase-catalysed transformation of ε-CL (4) to PCL (5) is a well-
ation) and the organic phase (for in situ-product removal). known process albeit being reported at higher reaction temp-
Since the PDMS-membrane is permeable for substrate 2, it can eratures and in different solvents.14 After demonstrating that
diffuse at the beginning of the reaction along the concen- this enzyme-catalyzed ring-opening polymerization of ε-CL
tration gradient from the aqueous to the organic phase, thus also works at room temperature (25 °C) in methylcyclohexane
reducing the inhibition of the enzymes in the aqueous com- at 100 mM substrate concentration, we combined the biocata-
partment. Due to the conversion of cyclohexanol (2) to ε-CL in lytic double oxidation of cyclohexanol 2 into ε-CL (4) with
the aqueous compartment, the concentration of 2 decreases in situ extraction and polymerization of ε-CL to PCL (Fig. 3).
and 2 present in the organic compartment diffuses back to the We were pleased to find that under these conditions conver-
aqueous phase.
sion of cyclohexanol (2) into ε-CL species (monomer, dimer,
In the final step we envisioned an extension of this type of oligomer and PCL) is increased to 81% in comparison to the
in situ extraction towards the integration of a further reactive single aqueous system, which only gave 50% conversion for
step, namely a direct polymerization of ε-CL (4) to PCL (5) cata- the biocatalytic synthesis of ε-CL (4).
lysed by an immobilized lipase B from Candida antarctica
In contrast to the PDMS-system without polymerization,
(CAL-B). Thus, in a modular fashion this chemoenzymatic syn- however, (Fig. 2) conversion is decreased (81% versus 97%),
which might be related to the observation of a white thin layer
on the PDMS-membrane at the end of the reaction, indicating
a blocking or clogging up of the membrane by adsorbed oligo-
mers and polymers of ε-CL as well as the need for further
process optimization.
Conclusions
In summary, we disclosed a novel synthetic route for the pro-
duction of the polymer poly-ε-caprolactone based on a chemo-
enzymatic reaction sequence. Toward this end, an initial
hydrogenation of phenol to cyclohexanol was conducted, and
the resulting crude product was directly used without work-up
in a biocatalytic double oxidation towards ε-caprolactone by
means of an alcohol dehydrogenase and a monooxygenase. In
order to overcome product inhibition effects, an in situ-
product removal strategy via extraction of ε-caprolactone from
an aqueous reaction medium with an organic solvent in the
presence of a permeable polydimethylsiloxane-membrane was
applied. Furthermore, this in situ-product removal by extrac-
tion was combined with an enzymatic polymerization in the
organic phase at 25 °C utilizing the lipase CAL-B. The obtained
crude product contained a polymer fraction with a degree of
polymerization comparable to commercial PCL, thus reinfor-
cing the synthetic potential of this process concept towards
the petrochemical polymer poly-ε-caprolactone starting from
phenol through the combination of a metal-catalysed hydro-
genation and three enzymatic steps without the need for iso-
lation and purification of any of the intermediates.
Fig. 3 Whole-cell catalytic biotransformation of cyclohexanol to ε-CL
in a pure aqueous buffer system (blue) and to PCL in a PDMS-thimble
system (orange). The compartment inside the PDMS-thimble contains
the aqueous solution and is selectively separated by the PDMS-mem-
brane from methylcyclohexane (outside the thimble; for in situ extrac-
Acknowledgements
tion of ε-CL). In situ extraction is coupled with CAL-B-catalyzed
polymerization of ε-CL towards PCL in the organic phase. The crude
product contains a monomer (ε-CL), a dimer as well as oligomers and
polymers. This polymer fraction has a degree of polymerization compar-
The authors thank the German Federal Environmental
Foundation (Deutsche Bundesstiftung Umwelt, DBU) for
generous support within the DBU-network “ChemBioTec” of
the funding priority “Biotechnology” (project AZ 13268-32).
able to commercial PCLs (see gel permeation chromatograms).
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