Development of a lipase-mediated epoxidation process for monoterpenes in choline chloride-based deep eutectic solvents

Article abstract of DOI:10.1039/c7gc01127j

Chemical syntheses in contemporary process industries today are predominantly conducted using organic solvents, which are potentially hazardous to humans and the environment alike. Green chemistry was developed as a means to overcome this hazard and it also holds enormous potential for designing clean, safe and sustainable processes. The present work incorporates the concepts of green chemistry in its design of a lipase-mediated epoxidation process for monoterpenes; the process uses alternative reaction media, namely deep eutectic solvents (DESs), which have not been reported for such an application before. Choline chloride (ChCl), in combination with a variety of hydrogen bond donors (HBD) at certain molar ratios, was screened and tested for this purpose. The process was optimized through the design of experiments (DoE) using the Taguchi method for four controllable parameters (temperature, enzyme amount, peroxide amount and type of substrate) and one uncontrollable parameter (DES reaction media) in a crossed-array design. Two distinct DESs, namely glycerol:choline chloride (GlCh) and sorbitol:choline chloride (SoCh), were found to be the best systems and they resulted in a complete conversion of the substrates within 8 h. Impurities (esters) were found to form in both the DESs, which was a concern; as such, we developed a novel minimal DES system that incorporated a co-substrate into the DES so that this issue could be overcome. The minimal DES consisted of urea·H₂O₂ (U·H₂O₂) and ChCl and exhibited better results than both the GlCh and SoCh systems; complete conversions were achieved within 2 h for 3-carene and within 3 h for both limonene and α-pinene. Product isolation with a simple water/ethyl acetate based procedure gave isolated yields of 87.2 ± 2.4%, 77.0 ± 5.0% and 84.6 ± 3.7% for 3-carene, limonene and α-pinene respectively.

Full text of DOI:10.1039/c7gc01127j

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Page 1 of 22
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Development of a LipaseꢀMediated Epoxidation Process for Monoterpenes in Choline Chlorideꢀ
Based Deep Eutectic Solvents
a
b,c
a,b,d*
Sumanth Ranganathan , Sandra Zeitlhofer and Prof. Volker Sieber
aꢀ Technical University of Munich, Chair of Chemistry of Biogenic Resources, Schulgasse 16,
Straubing 94315, Germany.
bꢀ Fraunhofer Institute of Interfacial Engineering and Biotechnology (IGB), Bioꢀ, Electroꢀ and
Chemo Catalysis (BioCat) Branch, Schulgasse 11a, Straubing 94315, Germany
cꢀ University of Applied Sciences Upper Austria, School of Engineering/Environmental Sciences,
Stelzhamerstrasse 23, Aꢀ4600 Wels, Austria
dꢀ Technical University of Munich, Catalysis Research Center (CRC), ErnstꢀOttoꢀFischer Straße 1,
Garching 85748, Germany
*
ꢀ Corresponding author
Email: sieber@tum.de
Tel.: +49ꢀ9421ꢀ187ꢀ300

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ABSTRACT
Chemical syntheses in contemporary process industries today are predominantly conducted using organic
solvents, which are potentially hazardous to humans and the environment alike. Green chemistry was
developed as a means to overcome this hazard and it also holds enormous potential for designing clean,
safe and sustainable processes. The present work incorporates the concepts of green chemistry in its
design of a lipaseꢀmediated epoxidation process for monoterpenes, the process uses alternative reaction
media, namely deep eutectic solvents (DESs), which have not been reported for such an application
before. Choline chloride (ChCl), in combination with a variety of hydrogen bond donors (HBD) at certain
molar ratios, was screened and tested for this purpose. The process was optimized through the design of
experiments (DoE) using the Taguchi method for four controllable parameters (temperature, enzyme
amount, peroxide amount and type of substrate) and one uncontrollable parameter (DES reaction media)
in a crossedꢀarray design. Two distinct DESs, namely glycerol:choline chloride (GlCh) and
sorbitol:choline chloride (SoCh), were found to be the best systems and they resulted in a complete
conversion of the substrates within 8 h. Impurities (esters) were found to form in both the DESs, which
was a concern; as such, we developed a novel minimal DES system that incorporated a coꢀsubstrate into
the DES so that this issue could be overcome. The minimal DES consisted of urea·H O (U·H O ) and
2
2
2
2
ChCl and exhibited better results than both the GlCh and SoCh systems; complete conversions were
achieved within 2 h for 3ꢀcarene and within 3h for both limonene and αꢀpinene. Product isolation with a
simple water/ethyl acetate based procedure gave isolated yields of 87.2 ± 2.4 %, 77.0 ± 5.0 % and 84.6 ±
3.7 % for 3ꢀcarene, limonene and αꢀpinene respectively.
Keywords: deep eutectic solvents, design of experiments, green chemistry, lipase, monoterpene epoxide

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1
Introduction
1
ꢀ3
The utilization of renewable resources is one of the twelve principles of green chemistry and in addition
to the improved sustainability afforded by using renewables, the molecules obtained offer novel
functionalities. Recent examples of this include the synthesis of new bioꢀbased materials such as
4
5
polycarbonates from terpenes or furanic polyesters from sugars . Mere utilization of renewable
feedstock, however, is not necessarily a more sustainable practice than using non renewable ones as it is
still important that also other rules of green chemistry are followed; for example, green chemistry requires
1
ꢀ3
toxic and harmful chemicals to be used only sparingly, if at all, in chemical processes . To achieve this,
6ꢀ8
9, 10
solventꢀfree synthesis or “green” reaction media such as supercritical (SC) fluids
or ionic liquids
1
1ꢀ13
7, 9, 13
(
IL)
can be used. However, these systems often are impractical for chemical synthesis
. An
1
4ꢀ16
alternative approach is the use of deep eutectic solvents (DESs) . A variety of chemical reactions that
use DESs as reaction solvents have already been reported with subject areas ranging from
17, 18
19ꢀ21
22ꢀ25
electrochemistry
and organic syntheses
to enzymatic reactions
.
A good source of renewable feedstock is the secondary plant metabolite called terpenes that are
2
6, 27
accumulated in large quantities as byꢀproducts in the pulp, paper and fruit industries
. Terpenes are
excellent precursors for the flavor, fragrance and fine chemical industries in either a functionalized or a
nonꢀfunctionalized form. A specific functionalization, namely epoxidation, is instrumental in making
terpene epoxides useful precursors for the production of diols, alcohols, ketones and as of late, monomers
4, 28ꢀ32
for polymers
. Epoxides can be produced by different chemical means, including enzymatic
3
3ꢀ41
41
apporaches . Björkling et al. pioneered work on enzymeꢀmediated epoxidations in 1992 when they
used a lipase (Candida antartica lipase B (CALB)) in the presence of an organic solvent, carboxylic acid
and aqueous hydrogen peroxide (H O ) to form peroxycarboxylic acid, which was able to epoxidize
2
2
39
alkenes through the Prilezahev reaction . It is important to note that the enzyme does not catalyze the
epoxidation itself but provides efficient in situ formation of the oxidizing species, i.e. the
peroxycarboxylic acid.
41
Previously, we had used this technique to develop and optimize a lipase mediated epoxidation process
42
for monoterpenes . Although this process adhered to some principles of green chemistry, in that it
utilized renewable reactants and enzymes as catalysts; we also used toluene as the reaction medium;
which means the process cannot be considered as “green”.
This paper focuses on the development of a more sustainable or “greener” monoterpene epoxidation
process that adheres to the principles of green chemistry. To the best of our knowledge, this is the first
account that uses DES as solvent for lipase mediated epoxidation of monoterpenes. To begin with, we
tested an enzymeꢀmediated process both under solventꢀfree conditions and then in DESs as the reaction

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medium. After comparing these two approaches, we developed the process and optimized it for the DES
system. The process development stages consisted of two screenings, an optimization by design of
experiments (DoE) ꢀ Taguchi method, purification stage and a final scaleꢀup phase. We also wanted to
examine how using a DES as the reaction solvent affected the outcome (i.e., yield of the epoxide) of the
process as well as how other reaction parameters (i.e., substrate type, enzyme amount, temperature of
reaction and hydrogen peroxide) affected the outcome. We were subsequently able to develop a novel
DES mixture that could act as both the solvent and the coꢀsubstrate source in fast and efficient
epoxidations (Scheme 1)
Scheme 1: Development of the lipaseꢀmediated epoxidation process for various reactants (1aꢀ3a)
and their corresponding epoxides (1bꢀ3b), starting with the “ungreen” process utilizing toluene and
moving on to greener processes utilizing deep eutectic solvent (DESs) and solvent free conditions.
2
2
Results and Discussion
.1 Solvent free epoxidation system
The initial test of the solventꢀfree synthesis was performed using only terpene (3ꢀcarene (1a), limonene
2a) and αꢀpinene (3a)), octanoic acid, a peroxide source (aqueous (aq.) or urea (U) ·H O ) and CALB, as
(
2
2
specified in section 4.2.1.1. There were two distinct phases: a top organic phase that contained both the
monoterpene and octanoic acid and a bottom phase containing H O . A single point measurement at the
2
2

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end of 16 h revealed that 0.1 mmol 3ꢀcarene (1a) was totally converted to its corresponding epoxide
result not shown). Based on this result, we scaled up the process using a greater amount of the reactants
(
at 45 °C and 60 °C. The results are shown in Figure 1.
Figure 1: Conversion profile of 3ꢀcarene (diamond), limonene (square) and αꢀpinene (triangle) at
4
5 °C (a) and 60 °C (b). (Reaction conditions: 10 mmol monoterpene, 12.5 mmol aq.H O , 2.5 mmol
2 2
octanoic acid, 100 mg CALB (1670 PLU), 500 rpm)
At 45°C (Figure 1 (a)), conversions of 53.0 ± 0.8 %, 46.6 ± 1.8 % and 13.8 ±0.1 % were achieved after
0 h for 1a, 2a and 3a respectively. A second reaction was carried out using identical conditions, but the
2
reaction temperature was increased to 60 °C. At this temperature (Figure 1 (b)), a conversion of 100 %
was seen for 1a, whereas 2a and 3a yielded 82.8 ± 2.2 % and 5.5 ± 1.2 %, respectively. When the reaction
time was extended to 20 h, no increase in the conversion of 2a could be achieved. Only when adding
fresh enzyme after 6 h an increase in conversion was observed (data not shown), implying that
inactivation of the enzyme, possibly due to the high amount of octanoic acid in the reaction medium was
limiting.
More interestingly, the results of this experiment are different from those obtained in our previous work,
which had implied that the best results for the lipase epoxidation of monoterpenes in toluene were to be
4
2
achieved at a reaction temperature of 45 °C . Our present results also indicate that the sequence in which
the three substrates were oxidized, at both temperatures, was different from that reported by Bakhvalov et
4
3
al. in 2008 . The present work suggests that the oxidation follows the order 1a > 2a > 3a rather than 1a >

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3a > 2a. However, the published findings are for solventꢀbased oxidation reactions, which have better
heat and mass transfer conditions than solventꢀfree reactions. Additionally, each substrate behaves
differently as a solvent, which could have caused the variation in the oxidation pattern. Furthermore, the
42, 43
results in both of the previously published studies
were obtained using optimized conditions as
opposed to the single variable change technique used in the present study.
As mentioned earlier, two distinct phases were observed when aq. H O was used. Suspecting that the
2
2
water content may have interfered with the reaction, we conducted a third test using three different
temperatures and U·H O . On mixing all the reactants together, a single solid phase was obtained. After
2
2
20 h, a single point measurement was made and conversion of the monoterpenes was calculated using gas
chromatography–mass spectrometry (GC–MS), as shown in Figure 2.
Figure 2: Conversion of 3ꢀcarene (dotted) and limonene (vertical dashes) obtained using U·H O2
2
under solventꢀfree conditions at 40, 50 and 60 °C after 20 h. (The reaction conditions were 2 mmol
monoterpene, 2.5 mmol U·H O , 0.5 mmol octanoic acid, 100 mg CALB (1670 PLU), 500 rpm)
2
2

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Conversions of 85.3 ± 10.4 %, 73.4 ± 7.9 % and 84.5 ± 14.4 % were obtained for 1a and 86.2 ± 4.8 %,
2.2 ± 1.8 % and 75.8 ± 7.2 % for were obtained for 2a at 40, 50 and 60 °C, respectively after 20 h. There
6
was no conversion of 3a at any of the three tested temperatures. On a closer observation of Figure 2, it
can be seen that the results of the epoxidations at 40 and 60 °C were identical for 1a, but not for 2a.
However, an interesting phenomenon can be seen at 50 °C, for which the conversion was the lowest. This
may have occurred due to the difference in the solubility of the octanoic acid in each of the substrates (1aꢀ
3a).
Both the systems, i.e. aq. H O and U·H O , exhibited incomplete conversions of the starting materials
2
2
2
2
(
1aꢀ3a) with the exception of the reaction at 60 °C using aq. H O for 1a. If a process has to be developed
2 2
so that maximum conversion is obtained for all three substrates, optimization using, for example, the DoE
approach can be carried out. However, each substrate would act as its own solvent and any optimization
will be useful only for that particular substrate. Additionally, both the cases (i.e. aq. H O and U·H O )
2
2
2
2
had issues in terms of both handling and reproducibility (as evidenced by the high error percentage of the
tests); therefore, we shifted our focus from a solvent free system towards utilizing green reaction media,
namely DESs.
2.2 Conventional DES, first screening round
Because of the drawbacks experienced on using the solventꢀfree system (section 2.1), DESs were chosen
as a “green” alternative to carry out the lipase mediated epoxidation reactions. To determine the best DES
candidates, a twoꢀstep screening approach was used: an initial screening step to evaluate the fluidity of
the selected DES reactants and a second step was performed to evaluate their epoxidation of 1a. The
mixtures that were assumed to be suitable for the epoxidation process were chosen from the list published
15
by Russ and Koenig . The DES mixtures were prepared in ratios described in Table 2 according to the
procedure described in section 4.3. All the mixtures in Table 2 had been previously described by Russ and
15
Koenig apart from the 4ꢀhydroxy phenyl acetic acid (HPA) and ChCl mixture (Table 2, #3). This
particular chemical (HPA) was chosen because phenylacetic acid was described in the same work as
having a melting point of 25 °C in the same molecular ratio. Because both the chemicals are similar in
structure but for the presence of an additional OH group, this mixture was tested to see if a new DES
mixture could be formed in a similar temperature range.
From previous experience it has already been established that 45 ꢀ 60 °C is the ideal temperature for
4
1, 42, 44
performing lipase based epoxidation reactions
. Hence, only those DESs that were liquids at 60 °C
were selected for the second round of screening. The DES mixtures from Table 2 were heated to 100 °C

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and cooled to 60 °C before the samples were visually examined for fluidity; the results are given in Table
. HPA, Lꢀ(+)ꢀtartaric acid, Lꢀglutamic acid and Dꢀglucose in combination with ChCl did not yield a
2
liquid at 60 °C and malonic acid with ChCl (1:1) only yielded a very viscous liquid that could not be
stirred. As a result, these mixtures were not used for the epoxidation reaction.
15
Literature suggested that the selected mixtures are supposed to yield liquids at temperatures much lower
than those tested in this work. Meng et al. suggested that the presence of moisture can interfere with the
hydrogen bonding between DES components (urea and ChCl), which would lead to increased melting
45
temperatures . Working on the assumption that this phenomenon could be extended to other DES
mixtures, the individual DES components (that were not liquid) were dried under vacuum and tested
again; no changes in their behaviors were observed. Since the DES mixtures mentioned above (HPA,
tartaric acid, glutamic acid and Dꢀglucose in combination with ChCl) did not form liquids and this step
was a mere screening round, they were omitted from the second round of screenings and were not
investigated further.
2.3 Conventional DES, second screening round
The eight successful liquid DES mixtures from the previous screening round were screened for
epoxidation activity, as described in section 4.4. The conversions after 24 h of reaction time are given in
Table 2. It can be inferred that the sugar and sugar alcohol systems were the ones that performed best. For
the carboxylic acid systems (Table 2, #1 & 2), no additional peroxy acid generator, i.e. octanoic acid, was
added. These two reactions yielded minimal conversion, which may have been due to the polar nature of
both these acids, as the polarity of a carboxylic acid increases with a decrease in its aliphatic chain.
1
5
However, the melting temperature required to produce a DES mixture also increases ; as a result, these
mixtures were not tested. In the case of urea: ChCl, it could be inferred that the combined effect of urea as
the HBD and the additional U·H O could have led to the inactivation of the lipase after a certain amount
2
2
4
6
of time. Because urea at a concentration of 6 M is known to be a denaturant of enzymes , we assumed
that this could be a reason for the reduced conversion. In order to test this, the lipases were washed three
times with water to remove the residual urea and then with ethanol to remove any terpene or terpenoid
impurity present. The reactions were then repeated and the conversion was either less than the previous
occasion or there was no conversion observed at all.
Considering the alcohol HBDs in combination with ChCl, we found the glycerol system (81.1 %) yielding
a better conversion than ethylene glycol (57.5 %). A suitable explanation for this behavior could be
4
7
obtained from the work of Rengstil et al that described the fluidity of a DES system to be directly

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proportional to the number of hydrogen bonds on offer from a HBD. As the conversion obtained for the
ethylene glycol DES mixture was below 80 %, it was not used for the optimization round. The fructose
based DES yielded a dark brown mixture and was not as stable as the sugar alcohols, hence it was also
not included in the optimization step. Lastly, because sorbitol is a cheaper resource than xylitol, it was
preferred for use in the optimization phase. As a result, only glycerol (GlCh) and sorbitol (SoCh) DES
mixtures were considered further.
2.4 Conventional DESꢀ optimization using the Taguchi method
The lipaseꢀmediated epoxidations were optimized for the GlCh and SoCh systems using the Taguchi
crossed array method. A detailed explanation of the choice of parameters for this optimization, the theory
,
48ꢀ53
behind the Taguchi method and the signal to noise ratio can be obtained from literature
. All reactions
were performed in the order as described in the Supplementary section (once for each of the systems, i.e.
GlCh and SoCh) in triplicate. Minitab (version 17) software was used to analyze the results. The response
variable used was the conversion of the monoterpenes (1a–3a) to their corresponding epoxides (1b–3b).
The results of the optimizations are given in detail in the Supplementary information.
The optimized set of parameters for maximum conversion of 1 mmol of terpene was similar for both
DES Systems used GlCh (5 mmol ChCl, 10 mmol Glycerol) and SoCh (5 mmol ChCl, 5 mmol Sorbitol):
4
mmol U·H O , 100 mg lipase at 40° C to 50 °C. Independent of the DES used, the conversion is more
2 2
efficient for 1a and 2a. Decreasing the amount of enzyme to 75 mg only slightly decreased the amount of
conversion. SoCh was found to work slightly better at lower temperatures (40 °C). Interestingly, a strong
dependence on the amount of U·H O was found for the conversion amounts of the two DES systems.
2
2
2.5 Evaluation of the substrate range in the GlCh and SoCh systems
After the optimal conditions for the processes, i.e. the GlCh and SoCh systems, were identified, two
additional substrates, camphene (4a) and 1ꢀdodecene (5a), were tested to verify the range of the DESs. 5a
was tested because it is a monoterpene and 4a was tested to verify if the process could be extended to the
terminal double bond of linear olefins as well (Scheme 2).

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Scheme 2: Lipaseꢀmediated epoxidation of camphene (4a) and 1ꢀdodecence (5a) to their
corresponding epoxides (4b and 5b) using the optimized set of parameters for the GlCh and SoCh
systems.
All reactions were performed with the optimized set of conditions described in section 2.4. The results for
the new substrates in addition to those tested in the GlCh system are shown below in Figure 3.
Figure 3: Conversions obtained for 1ꢀdodecene (circle), αꢀpinene (square), camphene (x),
limonene (triangle) and 3ꢀcarene (diamond) over time using the optimized GlCh system.
After 8 h, 1a and 2a were almost fully converted to their corresponding epoxides (1b and 2b), as
predicted by the DoE. 3a and 4a were approximately 83ꢀ88% converted and only 35% of 5a was
converted to 1ꢀdodecene epoxide (5b) after 8 h with 69% being converted after 24 h. Although increased
reaction times may ultimately improve the conversion, we did not test for this.
Similar tests were also performed for the SoCh system and the results are shown in Figure 4; similar
findings were obtained. The conversions of 1a and 2a were 100% and that of 3a was approximately 63 ꢀ

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70% after 8 h. However, 4a had a slightly lower conversion of 75 ꢀ 83% in the SoCh system than in the
GlCh system. Sampling proved to be more difficult for the SoCh system than for the GlCh system. This
was because of the separation of phases (in the reaction vessel), which took longer for the SoCh system
compared to the GlCh system. 5a had a conversion of 55 ꢀ 65 %, which was surprising given that GlCh
system had a conversion of 25 ꢀ 35% for this substrate. We assume that the viscosity of the SoCh system
played a major role in this difference. A possible explanation is that sorbitol and ChCl may have formed a
dynamic DES system, wherein U·H O and octanoic acid might have been dissolved better (than the
2
2
GlCh system) leading to a faster peroxycarboxylic acid formation, resulting in a faster epoxidation
process.
Figure 4: Conversion obtained for 1ꢀdodecene (circle), αꢀpinene (square), camphene (x), limonene
(
triangle) and 3ꢀcarene (diamond) over time using the optimized SoCh system.
One major drawback of both the systems was the formation of caprylate esters of both glycerol and
sorbitol, which we detected using GCꢀMS (Supplementary Information). To produce pure epoxides (1bꢀ
5b) and avoid the formation of esters, we decided to shift the search toward DES mixtures that did not
contain any alcohol groups. This led to the development of the “minimal” DES system which consisted of
ChCl: U·H O that was to be used as both the peroxide source and the solvent.
2
2
2.6 Minimal DES results
We already demonstrated that the urea: ChCl (Table 2, # 9) system was liquid at the desired temperature,
i.e. 60 °C and yielded a conversion of 67 %. We therefore elected to use this system, albeit with a small

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modification: U·H O was used instead of urea for a novel DES to be formed. Doing so meant that
2
2
additional amounts of U·H O did not need to be added, as the compound already contains urea for DES
2
2
formation and the H O needed for epoxidation. This method was used along with the same reaction
2
2
conditions outlined in section 4.6 and the epoxidation was successful within 2 h for 1a and 2a, whereas it
took 3 h for 3a; this is shown both in Figure 5 and also and in Table 5 of the Supplementary Information.
It can be seen that after 2 h, 1a was completely converted to 1b, 2a was 99 ± 1 % and 3a was converted to
92 ± 6%. After 3 h, all the samples were completely converted to their epoxides.
Figure 5: Conversion profile of 3ꢀcarene (diamond), limonene (square) and αꢀpinene (triangle)
using the ChCl:U·H O DES mixture
2
2
This surprisingly good result could have been due to the urea and the ChCl forming a proper DES, with
the remaining H O being dissolved in the DES. This resulted in the educts and the peroxy acid generator
2
2
having better solubility, which led to faster reaction kinetics. This makes this reaction medium, which was
the simplest of all of the ones tested, the most effective one as well. In fact, it performed even better than
4
2
the toluene system that we had previously developed . It should be noted that this process itself was not
optimized using the Taguchi method, but the results of the previous optimizations were used here. The
epoxides produced were then purified according to the procedure described in section 4.8.
To analyze the purity of epoxide 1b, we carried out GCꢀMS and nuclear magnetic resonance (NMR)
analyses. As described above, in the samples from the GlCh and SoCh systems, esters formed between
octanoic acid and the alcohol groups of the DES was detected as impurity peaks in the GCꢀMS. No such
peak was present when the synthesis was performed in U·H O (Supplementary Information). In theory
2
2
the cholinium species could also lead to side product formation as it also contains an alcohol group. Since

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the GCꢀMS exhibited no impurities (Supplementary Information, Figure 6), we performed an additional
NMR analysis of purified 1b and compared the spectra of the samples from the two different systems.
The chemical shift presents the spectrum for 1b from the GlCh system (cyan) and ChCl:U·H O system
2
2
4
2, 44
(
red) (Figure 6). The NMR shifts of 1b match to the ones reported in literature
however, that peaks that correspond to octanoic acid esters at around 0.9, 1.3 and 2.3 ppm, are present in
b from the GlCh system, while these peaks are absent in 1b from the ChCl:U·H O system. Apparently,
. It can be seen,
1
2
2
lipase based esterification of choline with CALB is less efficient, possibly due to the positive charge of
the molecule, leading to decreased side product formation. We can therefore conclude that the
ChCl:U·H O2 system is much more efficient in producing epoxides in a purer form than the GlCh or
2
SoCh systems.
Figure 6: NMR spectrum of 3ꢀcarene epoxide produced by GlCh (top) and ChCl:U
systems.
·
H O (bottom)
2 2
2.7 Product purification and isolated yields
The utilization of DES has implications for product purification. The low solubility of DES in organic
solvents can be exploited for a simple extraction process. Accordingly, nꢀhexane was first used as the
extraction solvent as described in detail in section 4.8.10. Isolated yields close to 90 % could be obtained
(
Table 1). However, the utilization of nꢀhexane counteracts the green principles of the process as it is

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5
4
considered a harmful organic solvent . An effective replacement for the extraction solvent was to be
investigated. The high water solubility of the DES constituents combined with the low water solubility of
the products actually might allow a water based extraction process. Hence, a new water based purification
scheme was developed. Water indeed dissolves the DES and three phases appear ꢀ the upper organic
phase with the product and octanoic acid, the middle phase with lipase beads and a lower DES phase that
can be discarded. The octanoic acid in the organic phase could then be deprotonated and transferred to the
aqueous phase yielding pure epoxide as an upper phase. However, this led to the loss of terpene epoxide
on the walls of the separating funnel, due to the work in small scale. To solve this issue, we used ethyl
acetate in combination with water in order to facilitate better separation of the DES and the organic
phases. On using the protocol described in 4.8.2, we were able to isolate the products with relative ease
and the results obtained are shown below in Table 1. It can be seen that the water and ethyl acetate
purification procedure is equal to or even slightly better than the nꢀhexane process.
Table 1: Comparison of isolated yields (%) of terpene epoxides (1bꢀ3b) obtained on using the nꢀ
hexane and water + ethyl acetate processes.
S.No. Product
Isolated yield (%) obtained on Isolated yield (%) obtained on
using nꢀhexane
89.8 ± 5.9
using water/ethyl acetate
87.2 ± 2.4
1
1b
2
3
2b (70 %), 2c (30 %)
74.0 ± 4.5
80.4 ± 7.0
77.0 ± 5.0
3b
84.6 ± 3.7
3
Conclusions
This work presents the epoxidation of monoterpenes under solventꢀfree conditions. Owing to the
incomplete conversion of reactants, with the exception of 1a, it can be inferred that individually tailored
optimizations are necessary for each monoterpene. To overcome these issues, DES, which is considered a
green reaction medium, was used to epoxidize monoterpenes. Two of these systems, i.e. GlCh and SoCh,
were successful in yielding complete conversions of the starting material within 6ꢀ8 h. However, both
these systems produced ester impurities. To avoid this, a novel “minimal DES” consisting of ChCl and
U·H O was developed, which achieved a total conversion of the reactants within 2ꢀ3 h. We were able to
2
2
reduce the reaction time by a quarter using this new DES system. In addition to this, we developed a
purification procedure using water and ethyl acetate that enables a good recovery of terpene epoxides
whilst maintaining the green aspect. To summarize, we believe that this new system could inspire future
works in this field not just at the laboratory scale but also at the industrial scale.

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4
Materials and Methods
.1 Materials
4
All the materials in this study were used as purchased without further modification or purification steps.
42
1
b was produced in house and was used as an analytical standard. (+)ꢀlimonene (96%) was purchased
from Acros Organics, Germany. Toluene (≥ 99.9%) was purchased from Merck KGaA, Germany.
Gylcerol (≥ 99.5%) was purchased from Roth chemicals, Germany. Dꢀsorbitol (min. 99%) and sodium
hydroxide (min. 99%) were obtained from Applichem GmBH. 3ꢀcarene (≥ 90%), αꢀpinene (98%), 1ꢀ
dodecene (95%), camphene (95%), choline chloride (≥ 98%), D(ꢀ) fructose (≥ 99%), potassium
carbonate(≥ 99%), Lꢀ(+)ꢀtartaric acid (≥ 99%), laevulinic acid (99% FG), Malonic acid (99%), Octanoic
acid (98%), ureaꢀhydrogen peroxide (U·H O ) (97%), urea (molecular biology grade), xylitol (99 %) and
2
2
zinc bromide (98%) were bought from Sigma Aldrich, Germany. Ethyl acetate (LCꢀMS grade, min.
9.95%), aqueous hydrogen peroxide (aq. H O ) (35 %) and nꢀhexane (> 95%) were obtained from
9
2
2
Th.Geyer GmBH, Germany. Ethylene glycol (≥ 99.5%) was purchased from VWR chemicals, Germany.
The enzyme Candida antartica lipase B (CALB) was procured from two suppliers ꢀ Chiral Vision
(
IMMCALBꢀT2ꢀTXL, 15000 PLU/g) was used for optimization reactions and cꢀLEcta (CALB Immo
plus, 16700 PLU/g) was used for all the other reactions. Both the commercial CALB preparations used in
this work were immobilized covalently on to identical hydrophobic supports with a similar enzyme
loading. Moreover, previous tests performed showed no characteristic difference in reactivity (results not
shown).
4.2 Methods
4.2.1 Solvent free epoxidation systems
The tests for the solventꢀfree epoxidation systems were carried out using two different peroxide sources:
aqueous (aq.) H O and urea (U)·H O .
2
2
2
2
4
.2.1.1 Aq. H O2
2
An initial test was carried out to determine whether a solventꢀfree epoxidation was even possible for
monoterpenes; this was done using 2 mmol 1a, 2.5 mmol aq. H O (35%), 0.5 mmol octanoic acid,
2
2
100 mg (1670 PLU) CALB, 40 °C and 500 rpm for a duration of 16 h.

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The scaledꢀup version was carried out using 10 mmol monoterpene (1a, 2a and 3a), 12.5 mmol of aq.
H O (35%), 2.5 mmol of octanoic acid, 100 mg (1670 PLU) CALB, 45 and 60 °C and 500 rpm for a
2
2
duration of 20 h (45 °C) and 8 h (60 °C).
.2.1.2 U·H O2
4
2
The test was performed using 2 mmol monoterpene (1aꢀ3a), 2.5 mmol U·H O , 100 mg (1670 PLU)
2
2
CALB, 0.5 mmol octanoic acid, 40, 50 and 60 °C and 500 rpm for a reaction time of 20 h.
4.3 Conventional DES, first screening round
1
5
Several DES mixtures described by Russ and Koenig were prepared with the assumption that they
would be appropriate reaction media for the lipaseꢀmediated epoxidation reaction. ChCl was used as the
halide salt and different HBDs at certain ratios (described in detail in Table 2) were used to form the DES
mixtures. For the preparation of the DES, ChCl and the corresponding HBD were carefully weighed into
an empty 20 ml reaction vessel. The vessel was then heated to 100 °C for 120 minutes, after which the
samples were cooled to 60 °C. The fluidity of the DES mixture was visually examined and noted. The
samples that were liquid at 60 °C were then used as the reaction media in the second round of the
screening process.
Table 2: List of HBDs and ChCl screened as DES for the first round of screening. ChCl:HBD are
given in molar ratios. T °C refers to the melting point of the mixtures and RT refers to room
1
5
temperature as described by Russ & Koenig . Conversion refers to the amount of 3ꢀcarene
converted to its respective epoxide during the second round of screening.
S.No.
HBD
Type
ChCl :
HBD
T °C
Fluidity at Conversion (%)
60 °C
1
2
3
Valeric acid
Carboxylic acid
1:2
RT
Yes
Yes
No
No conversion
Laevulinic acid Carboxylic acid
1:2
1:2
RT
17.4
NA
4ꢀhydroxy
phenyl acetic
acid
Carboxylic acid
No
data
4
Malonic acid
Dicarboxylic acid 1:1
10
Yes*
NA

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5
6
7
8
9
1
1
1
1
*
L (+) tartaric
acid
Dicarboxylic acid 2:1
47
13
ꢀ40
ꢀ20
12
5
No
NA
NA
81.1
57.5
66.9
100
NA
100
100
LꢀGlutamic
acid
Amino acid
Alcohol
1:2
1:2
1:2
1:2
1:2
1:2
1:1
1:1
No
Glycerol
Yes
Yes
Yes
Yes
No
Ethylene glycol Alcohol
Urea
Amide
0
1
2
3
DꢀFructose
DꢀGlucose
DꢀXylitol
DꢀSorbitol
Sugar
Sugar
14
RT
RT
Sugar alcohol
Sugar alcohol
Yes
Yes
Was liquid, but highly viscous; NA ꢀ not applicable
4.4 Conventional DES, second screening round
All the DES mixtures that were liquids at 60 °C ( Table 2) from the previous screening round were used
as the reaction media for the lipase mediated epoxidation of 1a. A typical lipase reaction screening
experiment consisted of 1 mmol 1a, 0.25 mmol octanoic acid, 3 mmol U·H O , 100 mg (1670 PLU)
2
2
CALB and the liquefied DES mixtures from the first screening round. The reaction was carried out at
0 °C and 500 rpm in an oil/sand bath. In order to have sufficient reaction medium for the epoxidation
6
reactions, the DES mixtures were prepared at an increased factor of five whilst maintaining the same
molecular ratio (For example: 5 mmol ChCl with 10 mmol glycerol). A single point measurement was
taken at the end of 24 h to determine the conversion of 1a to 1b, and the measurement is described in
Table 2.

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4.5 Optimization
The lipaseꢀmediated epoxidation process was optimized using the DoE Taguchi method. The theory
4
2
behind this method has already been described in detail in our previous work as well as in various other
48ꢀ52
studies , as such, it will not be discussed in the present study. Although this method was used in the
present study, DESs were used instead of organic solvents. The parameters chosen and the levels used are
given in Supplementary Information. The optimizations were performed using two L orthogonal arrays
9
(
Supplementary Information) and the performance criterion used was “larger is better”. Each row in an
array corresponds to the combination of parameters at their respective levels. The constant parameters
used in the process were: 500 rpm mixing and the source of lipase (CALB from Chiral Vision with a
loading of 15000 PLU/g). The reactants were mixed in the order described in the Supplementary
information. The arrays for the trials and the analysis of the results were generated using Minitab
(
version 17) software.
4.6 Minimal DES mixture
ChCl (7.5 mmol) and U·H O (15 mmol) were mixed at room temperature between 45 min to 1 h with a
2
2
magnetic stirrer. The resultant fluid mixture was then used as both a solvent and a peroxide source for the
lipaseꢀmediated epoxidation reaction. The following reaction conditions were used: 5 mmol monoterpene
(
1aꢀ3a), 100 mg (1670 PLU) CALB, 1.25 mmol octanoic acid, 50 °C and 500 rpm.
4.7 Analytics and sampling
The GCꢀMS and NMR settings, heating profile of gas chromatography and mass spectrometry details, in
addition to the retention times of the reactants and products have already been described in our previous
4
2, 44
works
. The sampling was performed as follows (as DESs do not follow the traditional rules of
solvents, this study utilized different sampling techniques to analyze the compounds):
•
Up to 1 mmol of the starting material: 2 µl of organic phase (DES and enzyme free) was mixed
with 198 µl nꢀhexane. 10 µl of this sample was then transferred to 990 µl ethyl acetate
Up to 10 mmol starting material: 2 µl of organic phase was added to 998µl ethyl acetate
Up to 100 mmol of starting material: 1 µl of organic phase was mixed with 999 µl ethyl acetate
•
•
The samples were then subjected to GCꢀMS measurements. For the NMR measurements, 20 µl of pure
1
epoxide was mixed with 600 µl deuterated chloroform (CDCl ) and the sample was measured with H
3
proton NMR.

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4.8 Purification procedure
Terpene epoxides (1b, 2b, 2c and 3b) were produced in the minimal DES setup as mentioned in 4.6 and
4
2
was purified using an adapted version of our previous work . The purification procedure was developed
after an initial screening step (detailed description of the development is described in the supplementary
information). Two purification processes were tried for the effective recovery of the terpene epoxides
(
1bꢀ3b) in triplicates.
4
.8.1 Purification using nꢀhexane
The first method that was tried used nꢀhexane as the extraction solvent to extract the nonpolar fractions
epoxides (1aꢀ3b) and octanoic acid). This mixture was vortexed for 30 seconds to 1 minute. This mixture
(
was then cooled down to ꢀ20 °C for a time period of 1ꢀ2 h, which yielded three phases: a top organic
phase; a middle phase containing the lipase and a bottom DES phase (Supplementary information, SI
figure 3). The top organic phase was decanted and if necessary filtered ꢀ when lipase beads were found in
the organic phase. The organic phase was washed 3ꢀ5 times with 5 ml saturated sodium bicarbonate
solution (NaHCO ) for complete removal of octanoic acid. The organic phase was then dried using
3
anhydrous sodium sulphate (Na SO ). The nꢀhexane was then removed using vacuum distillation,
2
4
following which, the acid free epoxide was weighed and the isolated yield of the process was calculated.
.8.2 Purification using water and ethyl acetate combination
4
Owing to the harmful nature of the nꢀhexane and in the interest of making the process greener, the
following protocol was adapted. First, 10 mass equivalents of distilled water was added to the DES
mixture. This mixture was then vortexed at maximum speed for 30 s to 1 min. To this mixture, 10 ml of
ethyl acetate was added and vortexed for 30s to 1 min. This mixture was then transferred to a separating
funnel and 20 ml of saturated sodium bicarbonate (NaHCO ) solution was added to this mixture. The
3
organic phase was retained while the aqueous phase was discarded. This was repeated till the octanoic
acid was completely neutralized. The organic phase was then dried using anhydrous sodium sulphate
(
Na SO ) as before. The excess ethyl acetate was then removed using vacuum distillation, the terpene
2 4
epoxide (1bꢀ3b) weighed and the isolated yield of the process calculated.
Acknowledgments
We gratefully acknowledge the financial support of “Bayerisches Staatsministerium für Wirtschaft und
Medien, Energie und Technologie”. Additionally, we would like to thank Ms. Melanie Iwanow, Mr. Paul
Stockmann and Dr. Luciana Vieira for the theoretical and practical inputs that were helpful in completing
the paper in its present form.

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