Assessing biocatalysis using dihydrolevoglucosenone (Cyrene) as versatile bio-based (co)solvent

Article abstract of DOI:10.1016/j.mcat.2020.110813

An emerging biogenic aprotic solvent is the cellulose-derived dihydrolevoglucosenone, or 6,8-dioxabicyclooctanone (Cyrene). This paper explores the use of Cyrene in lipase-catalyzed biotransformations, both in aqueous solutions – as co-solvent – as well a

Full text of DOI:10.1016/j.mcat.2020.110813

Molecular Catalysis 485 (2020) 110813
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.elsevier.com/locate/mcat
Assessing biocatalysis using dihydrolevoglucosenone (Cyrene™) as versatile
bio-based (co)solvent
a,
b,
Nadia Guajardo *, Pablo Domínguez de María *
a
Programa Institucional de Fomento a la Investigación, Desarrollo e Innovación, Universidad Tecnológica Metropolitana, Ignacio Valdivieso 2409, San Joaquín, Santiago,
Chile
b
Sustainable Momentum, SL. Av. Ansite 3, 4-6. 35011, Las Palmas de Gran Canaria. Canary Is. Spain
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cyrene
Esterification
Lipases
Biocatalysis
Biogenic solvents
An emerging biogenic aprotic solvent is the cellulose-derived dihydrolevoglucosenone, or 6,8-dioxabicy-
clooctanone (Cyrene™). This paper explores the use of Cyrene in lipase-catalyzed biotransformations, both in
aqueous solutions – as co-solvent – as well as non-conventional media for synthesis. Cyrene is useful as organic
solvent for lipophilizations using benzoic acid and glycerol as a model system for substrates with unpaired
solubilities. The immobilized lipase B from Candida antarctica is active in Cyrene, yielding up to ∼ 10 g product
−1
L
. Interestingly, crosslinked aggregates immobilized lipases (CLEA) remain significantly stable in Cyrene,
enabling its use along several catalytic cycles. Cyrene is highly hygroscopic and forms geminal-diol structures
with water, leading to solvent mixtures with a (tailored) gradient of polarities, what may be promising for
biocatalysis in aqueous solutions. CALB displays hydrolytic reactions with different proportions of Cyrene as
cosolvent, and CLEA derivatives remain fully active over 6 cycles at 30 % v/v Cyrene.
1. Introduction
aprotic solvent which could replace traditional aprotic solvents such as
N,N-dimethylformamide (DMF) and N-methyl-2-pyrrolidone (NMP)
The quest of novel environment-friendly solvents is currently an
[21,22]. Cyrene can be derived from cellulose through pyrolysis [23]
and hydrogenation [24], or through a combination of both processes
[25]. Very recently, alkene reductases have been proposed for the final
step of the synthesis (double bond reduction) [26]. Cyrene has already
shown interesting applications in different areas of (synthetic) chem-
istry, as recently reviewed [21]. Importantly, a promising feature of
Cyrene is the fact that subsequent chemical derivatizations can be en-
visioned (e.g. Grignard chemistry on the ketone group), leading to a
new family of (tuned) Cyrene-based solvents, with tailored properties
[27]. Furthermore, Cyrene is highly water-miscible, presumably due to
the formation of an equilibrium with its hydrate, forming a ketal, the
Cyrene geminal-diol (Fig. 1). This phenomenon occurs also with 1,2-
diols, allowing the formation of novel solvent types (the so-called Cy-
gnets solvents family) [27,28]. Remarkably, these in-situ created deri-
vatives may display a gradient of polarities depending on their pro-
portions, rendering tailored solvents that may dissolve a broad range of
organic molecules [28].
important trend in Sustainable Chemistry, as solvents impact sig-
nificantly in the ecological footprint of chemical processes [1–4]. An
increasing number of promising solvents are becoming available, like 2-
methyltetrahydrofuran (2-MeTHF) [5–8] cyclopentyl-methyl ether
CPME) [9–11], other biogenic alternatives (e.g. p-Cymene, etc.)
12,13], or even solvent-free processes. Solvents that may be versatile,
tuneable, and able to dissolve chemicals with unpaired solubilities are
particularly appreciated, as they may be adapted to many challenging
synthetic processes. Herein, Deep Eutectic Solvents (DES) are proto-
typical examples, as the combination of hydrogen bond donor and ac-
ceptors may lead to solvents with different physical-chemical properties
(
[
[
14–16]. Connected to solvents, the use of enzymes as catalysts re-
presents a valuable alternative for (sustainable) organic synthesis, with
remarkable successful examples at industrial scale [17–19]. Combining
environment-friendly solvents with biocatalytic reactions may become
quite synergistic, in particular when continuous processes are im-
plemented [20]. Identifying novel enzyme-compatible biogenic sol-
vents is thus of high interest.
The potential of the versatility of Cyrene to create a novel family of
biogenic and designed solvents – together with its high water-mis-
cibility – may be of high relevance for biocatalysis, as mixtures of
Cyrene-water (e.g. as co-solvent) with adapted properties can be
A
recent promising alternative is the use of dihy-
drolevoglucosenone, or 6,8-dioxabicyclooctanone (Cyrene™) as dipolar
⁎
Corresponding authors.
E-mail addresses: nguajardo@utem.cl (N. Guajardo), dominguez@sustainable-momentum.net (P. Domínguez de María).
https://doi.org/10.1016/j.mcat.2020.110813
Received 12 November 2019; Received in revised form 31 January 2020; Accepted 1 February 2020
2468-8231/ © 2020 Elsevier B.V. All rights reserved.

N. Guajardo and P. Domínguez de María
Molecular Catalysis 485 (2020) 110813
Fig. 1. Cyrene™ synthesis and some possibilities for derivatization to form a family of novel biogenic and tunable solvents [27,28].
envisaged. Moreover, Cyrene may be chosen in enzymatic synthetic
reactions as well – using it as organic solvent to create a non-conven-
tional media –, in which substrates with unpaired solubilities are em-
ployed. However, examples of using Cyrene in biocatalysis are still
scarce and rather discouraging [12,29]. The use of Cyrene as organic
media in the lipase-catalyzed esterification of 2-phenylpropionic acid
with ethanol did not lead to conversion, while more apolar solvents (e.g.
hexane or biogenic p-Cymene) did result in effective esterification [12].
Albeit, arguably, the low LogP of Cyrene (-1.52) [12] may become a
hurdle for lipase-catalyzed synthetic reactions, the poor synthetic re-
sults obtained might also be partly explained by the enzyme inactiva-
tion at high acidic concentrations (200 mM of phenylpropionic acid)
2. Materials and methods
2.1. Chemicals and materials
Cyrene™, benzoic acid and glycerol were purchased from Sigma and
the biocatalyst Novozym 435 was kindly donated by Blumos S.A
(Chile). All reagents were used without further purification.
2.2. Protocol for CLEAs preparation
Crosslinked aggregates of CALB (CALB-CLEA) were prepared by
mixing the liquid enzyme (2.5 mL) from the original preparation with
5 mM phosphate buffer pH 7 (2.5 mL; 1:1 v/v). This enzyme solution
[
12], which may have a more dramatic effect in (highly hygroscopic)
2
polar media than in apolar ones. Herein, a derivatization of the solvent
to create a more apolar system (see some potential options for Cyrene in
Fig. 1), [27,28] while keeping efficient unpaired solubilities, would be a
promising research line. A new family of biogenic (co)solvents would be
thus available for biotransformations in aqueous and in non-conven-
tional media.
was enriched by BSA in a mass ratio of enzyme/BSA = 2.5 until the BSA
dissolved completely by shaking gently to avoid generating foam. To
this enzyme/albumin solution, 10 mL of a saturated solution of am-
monium sulfate was added dropwise in a cold bath at 4 °C. To achieve a
complete precipitation of the proteins present, the solution was kept for
30 min. Then glutaraldehyde (25 % v/v) was added dropwise in a molar
This paper explores the use of Cyrene in biocatalysis, using it in
hydrolytic reactions (as co-solvent), as well as in non-conventional
media for lipase-catalyzed lipohilizations. Herein, benzoic acid and
glycerol are used as prototypical substrates with unpaired solubilities,
to render α-monobenzoate glycerol α-MBG (Fig. 2). Glycerol and ben-
zoic acid are esterified by using immobilized lipase B from Candida
antarctica (CAL-B). The α-MBG has applications as emulsifier agent
ratio of glutaraldehyde/enzyme = 0.027 (2 mL) and was allowed to
react with constant stirring at 300 rpm for 1.5 h at 4 °C. The resulting
suspension containing the CLEA was centrifuged at 5 °C for 10 min at
1
0,000 g and the supernatant was discarded. Later on, the CLEA was re-
suspended in 100 mM phosphate buffer pH 7 and was re-centrifuged at
0,000 g for 5 min, this operation was repeated three to four times until
the biocatalyst was washed. The resulting biocatalyst was stored at 4 °C
39].
1
[
[
30], plasticizer [31], as well as building block in pharmaceuticals
[
32–34]. Due to the unpaired polarities of the substrates, many solvents
have already been evaluated, leading to moderate-to-high conversions
over 60 %) [30,35,36]. A particularly relevant example is the use of
2.3. Enzyme activity determination
(
Deep Eutectic Solvents (DES), which are efficient media for these en-
zymatic reactions [37–40].
Enzymatic activity was determined by measuring the absorbance at
48 nm, produced by the release of p-Nitrophenol (p-NP) resulting from
3
Fig. 2. Lipase-catalyzed esterification of benzoic acid and glycerol to obtain α-MBG using Cyrene as reaction media.
2

N. Guajardo and P. Domínguez de María
Molecular Catalysis 485 (2020) 110813
the hydrolysis of 0.3 mM p-NPB (ε = 5.8 [Abs x L x mmol-1]) in 25 mM
phosphate buffer pH 7 at 30 °C. An international unit of activity (IU)
was defined as the amount of enzyme that hydrolyzes 1 μmol of p-NPB
per minute [39].
2.4. General procedure for esterification
The esterification was performed in a sealed reactor, and the mix-
ture (substrates and solvent) was shaken at 165 rpm at 60 °C. The re-
actor was loaded with the biocatalyst, and then the reaction started
when the reaction media was added. The media was prepared by
mixing the substrates (glycerol and benzoic acid) with the Cyrene™ at
60 °C for 40 min. The conversion was calculated according to Equation
(
1):
n
p
− npo
vs
⎡
⎤
Conversion(%) =
×
× 100
⎢
⎣
⎥
⎦
n
so
v
p
Where P = product; nPo = amount of product P at the start of the
reaction (mol); nSo = amount of substrate S at the start of the reaction
(
P S
mol); n = amount of product P at the end of the reaction (mol); v =
stoichiometric factor for substrate S; and v
product P.
P
= stoichiometric factor for
2.5. Operational stability
To determine the operational stability of the biocatalysts under re-
action conditions, recycling of the biocatalysts was performed in se-
quential batch reactions with the same amount of biocatalyst per vo-
lume of reaction media in each batch. After each batch, the biocatalyst
was recovered from the reaction medium by filtration for Novozym 435
and centrifugation at 10,000 rpm for CALB-CLEA and was washed three
times with 25 mM phosphate buffer pH 7. The biocatalysts were then
washed three times with 25 mM phosphate buffer pH 7.
Fig. 3. Effect of temperature (A) and biocatalyst loadings (B) on the ester-
ification of benzoic acid and glycerol in Cyrene. Reaction conditions: Figure A:
6 h of reaction, 100 mg of biocatalyst (Nov-435), 3 mL of reaction volume,
50 mM benzoic acid, 50 mM glycerol, different temperatures; Figure B: 24 h of
reaction, 3 mL of reaction volume, benzoic acid 10 mM, glycerol 200 mM, dif-
ferent biocatalyst loadings (Nov-435). With 10 mg CAL-B no conversion was
observed in the timeframe of the experiments.
2.6. Analytic method
firstly studied by using different conditions of temperature and bioca-
talyst loadings (Fig. 3). As observed, CALB can perform the lipophili-
zation in Cyrene, reaching full conversion when relatively high enzyme
loadings are added. The reaction reached higher rates when tempera-
ture increased up to 60 °C, as expected from an enzymatic process using
thermostable CALB as catalyst.
The reaction product was quantified by UHPLC (Altus A-30,
PerkinElmer with a diode array detector) with a Kromasil Eternity-2.5-
18 UHPLC column (100 × 4,6 mm I.D) at 273 nm, 30 °C and 0.5 mL/
min. Mobile phase A= H PO 0.1 % and mobile phase B = Methanol.
The gradients used are detailed in Table 1:
C
3
4
In a subsequent set of experiments, the substrate ratio was eval-
uated, by performing the reaction at different concentrations of benzoic
acid and glycerol (Fig. 4). To keep the acidic conditions low (up to
3
. Results and discussion
3.1. Effect of operational conditions in synthesis reaction
50 mM), increasing concentrations of glycerol were added to the reac-
tion system. As depicted in Fig. 4 at a ratio of 50/400 mM (benzoic acid
The solubility of benzoic acid and glycerol in Cyrene was firstly
assessed. In both cases, efficient dissolutions were observed up to 1 M of
each substrate, demonstrating the potential of Cyrene to serve as sol-
vent for challenging compounds with different polarities. It must be
noted that the intended enzymatic reaction had been successfully as-
sessed in dioxane as solvent as well, with excellent enantiomeric excess
(
(R)-enantiomer, > 99 %) [35]. It can be expected, thus, that dioxane
may share some analogous properties with Cyrene (highly water mis-
cibility, low LogP, polarity, etc.). Once the solubilities of benzoic acid
and glycerol were demonstrated in Cyrene, the enzymatic reaction was
Table 1
UHPLC gradients.
Time (min)
Mobile phase B (%)
0
30
44
30
30
1
1
2
4
4.2
0
Fig. 4. Effect of molar ratio of substrates on conversion in the esterification to
obtain α-MBG. Reaction conditions: 60 °C, 6 h of reaction, 100 mg of biocata-
lyst, 3 mL of reaction volume.
3

N. Guajardo and P. Domínguez de María
Molecular Catalysis 485 (2020) 110813
Fig. 5. Effect of water content on the conversion in the esterification to obtain
α-MBG. Reaction conditions: 60 °C, 6 h of reaction, 100 mg of biocatalyst (Nov-
Fig. 6. Kinetics of enzymatic esterification to produce α-MBG using Cyrene™as
a reaction media. Reaction conditions: 48 h of reaction, 333 mg of biocatalyst
4
35), 3 mL of reaction volume, 50 mM benzoic acid and 50 mM glycerol.
(Nov-435), 10 mL of reaction volume, molar ratio benzoic acid/ glycerol = 1/
20 and 60 °C. (●) 50 mM benzoic acid; (□) 10 mM benzoic acid.
/
glycerol) the conversion increased by approximately 6 % with respect
to the 50/50 mM ratio (benzoic acid / glycerol). Following classic
premises of an esterification, this may be due to the hygroscopic effect
of glycerol, which might reduce the available water, shifting the es-
terification to the product formation [29]. Nonetheless, in this case
another aspect that may play a role is the (partial) formation of a Cy-
gnet-type solvent between Cyrene and glycerol (see Fig. 1), which
might modify the apparent LogP of the newly solvent phase (Cyrene,
Cyrene geminal diol, Cygnet-glycerol), and would then create a more
hydrophobic media, which could be beneficial for the lipase synthetic
performance. The gradient-like distribution was evidenced by Costa
Pacheco et al. [27], with studies conducted with Cyrene-water mix-
tures. The enzymatic reactive system of our work appears to be more
complicated, as glycerol and benzoic acid are also present.
Triggered by the changes observed in the conversion, the effect of
water was subsequently assessed in the reaction. To this end, different
amounts of phosphate buffer (pH 7) were added to the Cyrene solvent,
in the range of 0–30 % (v/v) (Fig. 5). As depicted in Fig. 5, the reaction
displayed its optimum performance at an addition of 0–2 % phosphate
buffer (v/v) and decreased significantly at higher amounts. Several
aspects may be playing a role in these results. Firstly, the lipase may
need some water to keep its active structure, which would explain why
some water addition is beneficial (Fig. 4). Secondly, Cyrene is hygro-
scopic, and may probably absorb some of the added water, leading to
some geminal-diol solvent system, which may exert some beneficial
effect for the lipase (Fig. 1). Thirdly, a higher amount of water (> 2 %
v/v) may probably influence the esterification equilibrium, shifting it
back to the substrate side (glycerol and benzoic acid). The reaction
mechanism is an enzymatic hydrolysis reaction, in which the enzyme
catalyzes the breakdown of the linkages generating glycerol, benzoic
acid and water. With respect to Cyrene when forming derivatives with
water, it would act as water sink, suppressing (partly) the hydrolysis
reaction.
Fig. 7. Operational stability of the biocatalysts A) Nov-435 and B) CALB-CLEA.
Reaction conditions: 24 h reaction, 67 mg of biocatalyst, reaction volume 2 mL,
10 mM benzoic acid, 200 mM glycerol molar ratio substrates 1:20.
In a subsequent set of experiments, the reaction was studied in terms
of productivities, using different amounts of benzoic acid (10 and
reactors keeping the same amount of biocatalyst by volume of reaction
media. Before starting the next batch, the biocatalysts were washed
with 25 mM phosphate buffer pH 7. Remarkably, the Nov-435 deriva-
tive loses its operational stability after the first cycle, whereas the
CALB-CLEA remain active over several cycles, in which activity is
gradually lost (ca. half of it after 6 cycles). Apart from the expected
desorption of the enzyme and support dissolution in the Nov-435, the
low LogP of Cyrene may play a deleterious role [41]. Interestingly, the
CLEA derivatives seem to be more resistant to the solvent, presumably
due to their covalent bonding [37].
50 mM). Results are depicted in Fig. 6. At low concentrations of benzoic
acid (10 mM), full conversion was achieved in less than 48 h, ac-
−
1
−1
counting for a (still suboptimal) productivity of ∼3 g product L
d
.
Remarkably, the enzyme resulted active in the solvent system and with
5
0 mM of benzoic acid, and the enzymatic performance remained at a
−
1
constant rate for at least 50 h, accumulating 10 g product L
case.
in that
To further assess the operational stability in Cyrene, the enzymatic
esterification was carried out in sequential batch reactors, using the
commercially available, immobilized CAL-B (Nov-435), as well as
crosslinked aggregates of Lipase B from Candida antarctica (CALB-
CLEA) (Fig. 7). Operational stability was carried out in sequential batch
3.2. Cyrene™ as cosolvent in hydrolysis reaction
Once the potential of Cyrene as solvent for non-conventional media
4

N. Guajardo and P. Domínguez de María
Molecular Catalysis 485 (2020) 110813
other solvents in a screening of transaminases [42]. Likewise, a thor-
ough study on Cyrene, and how its tunability may change the LogP, and
make it more adaptable to lipase-catalyzed reactions, may be a further
line of research as well.
Table 2
Hydrolytic activity of Nov-435 and CALB-CLEA biocatalysts at different con-
TM.
centrations of Cyrene
CyreneTM concentration % (v/v)
Biocatalyst
Nov-435
Enzymatic activity (IU/gBiocat.)
0*
73,2
66,1
60,3
15,7
67
4. Conclusions
2
3
4
0
0
0
In summary, the use of Cyrene in biocatalysis, both as co-solvent for
CALB-CLEA
0*
hydrolytic reactions, as well as non-conventional media for biocatalytic
reactions has been assessed, using lipase-catalyzed lipophilization re-
actions involving substrates with unpaired solubility. Some lipase de-
rivatives (CLEA) display promising operational activities in both reac-
tion media. For instance, under reaction conditions of low benzoic acid
loadings, some buffer addition (up to 2 % v/v), full conversion in the
reaction is achieved. The achieved productivities are lower than those
observed in other systems (e.g. Deep Eutectic Solvents), but further
optimizations may be implemented, related to genetic design, enzyme
immobilization, substrate dose, and reactor set-up. Beyond this proof-
of-concept, Cyrene may become a very promising media for enzymatic
processes, not only related to lipases, but also extendable to other en-
zymatic systems. Actually, based on its outstanding versatility – en-
abling the formation of tailored solvents –, combined with its high
miscibility in water, what may allow its use as co-solvent as well, the
use of Cyrene in other enzymatic systems using ketoreductases, trans-
aminases, whole cells, etc., should be assessed. The fact that different
Cygnet-type solvents can be formed and create a gradient-like of solu-
bility properties of the system may in fact open exciting opportunities in
biocatalysis, introducing a new palette of bio-based solvents for enzy-
matic reactions.
2
3
4
0
0
0
50,7
47,6
28,8
*
The hydrolysis activity was determined in 25 mM of phosphate buffer pH 7.
CRediT authorship contribution statement
Nadia Guajardo: Conceptualization, Methodology, Data curation,
Investigation, Writing - original draft. Pablo Domínguez de María:
Conceptualization, Methodology, Data curation, Writing - review &
editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Fig. 8. Operational stability of the Nov-435 and CALB-CLEA biocatalysts in the
hydrolysis reaction of p-NPB in Cyrene 30 % (v /v). Reaction conditions:
Acknowledgements
0.3 mM of p-NPB, 10 min, and 30 °C.
NG acknowledges the financial support from Fondecyt N°11150215
(
Chile).
in biocatalysis was demonstrated, its assessment as co-solvent in aqu-
eous media for hydrolytic reactions was conducted. To that end, both
Nov-435 and CALB-CLEA biocatalysts were assessed in the hydrolysis of
p-nitrophenol butyrate (p-NPB) using different proportions of Cyrene™
as co-solvent (0–40 % v/v) (Table 2).
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