Lipase catalysed oxidations in a sugar-derived natural deep eutectic solvent

Article abstract of DOI:10.1080/10242422.2021.1913126

Chemoenzymatic oxidations involving the CAL-B/H₂O₂ system was developed in a sugar derived Natural Deep Eutectic Solvent (NaDES) composed by a mixture of glucose, fructose and sucrose. Good to excellent conversions of substrates like cyclooctene, limonene, oleic acid and stilbene to their corresponding epoxides, cyclohexanone to its corresponding lactone and 2-phenylacetophenone to its corresponding ester, demonstrate the viability of the sugar NaDES as a reaction medium for epoxidation and Baeyer-Villiger oxidation.

Full text of DOI:10.1080/10242422.2021.1913126

Biocatalysis and Biotransformation
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ibab20
Lipase catalysed oxidations in a sugar-derived
natural deep eutectic solvent
Martina Vagnoni, Chiara Samorì, Daniele Pirini, Maria Katrina Vasquez De
Paz, Dawit Gebremichael Gidey & Paola Galletti
To cite this article: Martina Vagnoni, Chiara Samorì, Daniele Pirini, Maria Katrina Vasquez
De Paz, Dawit Gebremichael Gidey & Paola Galletti (2021): Lipase catalysed oxidations
in a sugar-derived natural deep eutectic solvent, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2021.1913126
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2021.1913126
RESEARCH ARTICLE
Lipase catalysed oxidations in a sugar-derived natural deep eutectic solvent
ꢀ
Martina Vagnoni , Chiara Samorı, Daniele Pirini, Maria Katrina Vasquez De Paz, Dawit Gebremichael
Gidey and Paola Galletti
Department of Chemistry “Giacomo Ciamician”, University of Bologna, Ravenna, Italy
ABSTRACT
ARTICLE HISTORY
Received 7 January 2021
Revised 7 March 2021
Accepted 31 March 2021
Chemoenzymatic oxidations involving the CAL-B/H2O2 system was developed in a sugar derived
Natural Deep Eutectic Solvent (NaDES) composed by a mixture of glucose, fructose and sucrose.
Good to excellent conversions of substrates like cyclooctene, limonene, oleic acid and stilbene
to their corresponding epoxides, cyclohexanone to its corresponding lactone and 2-phenylaceto-
phenone to its corresponding ester, demonstrate the viability of the sugar NaDES as a reaction
medium for epoxidation and Baeyer-Villiger oxidation.
KEYWORDS
Chemoenzymatic oxidations;
green solvents; natural
deep eutectic solvents;
epoxidation; Baeyer-
Villiger; lipase
GRAPHICAL ABSTRACT
1. Introduction
that continuously forms the peracids through a lipase-
catalysed perhydrolysis of carboxylic acids or their
Oxidation reactions have always been a major area of
research due to their tremendous industrial applica-
tions. However, several oxidation processes present
sustainability issues from the point of view of oxi-
dants, catalysts and solvents used (Cavani and Teles
2009). For example, a peracid is often used as the oxi-
dising agent (Swern 1949), but transportation and
storage of organic peracids leads to significant safety
issues and costs; when achievable, molecular oxygen
or air are for sure the ideal oxidants, with hydrogen
peroxide as the second-best choice, in terms of atom
economy and applicability to various oxidation sys-
tems (Burek et al. 2019). In this context, enzymes, that
can work in sustainable solvents with mild oxidants,
can contribute to increase the greenness of the oxida-
tion reactions (Niku-Paavola and Viikari 2000;
Constable et al. 2007; Gorke et al. 2008; Kotlewska
et al. 2011; Silva et al. 2011; Hollmann et al. 2011;
€
esters (Bjorkling et al. 1990; Yadav and Devi 2002;
Busto et al. 2010). A broad range of hydrolases has
been investigated for the peracid formation and
among them the lipase B from Candida Antarctica
(CAL-B), immobilised onto an acrylic resin (Novozyme
435) is the most reactive (Ortiz et al. 2019). This sys-
tem has been successfully applied to both epoxida-
tions of alkenes (Prileshajev-epoxidation) and Baeyer-
Villiger (B-V) oxidations (Scheme 1) (Lemoult et al.
1995; Aouf et al. 2014).
Epoxides are fundamental intermediates in organic
synthesis but, despite their relevance, their industrial
synthesis is scarcely sustainable (both environmentally
and economically). Epoxidation of some natural prod-
ucts is industrially carried out by the Prileshajev-epoxi-
dation (epoxidation of an alkene with a peracid) using
either preformed or in-situ-generated short chain per-
€
oxy acids (Rusch Gen. Klaas and Warwel 1999; Hilker
_ _
Drozdz et al. 2015; Qin et al. 2015; Yin et al. 2015;
ꢁ
et al. 2001). Nevertheless, the need for a strong acid
to catalyse peroxy acid formation in this process can
result in unsatisfactory selectivity and undesiderable
Yang and Duan 2016; Garcıa et al. 2018).
In fact, a very interesting system for obtaining pera-
cids in situ is the chemoenzymatic system lipase/H2O2
CONTACT Martina Vagnoni
martina.vagnoni3@unibo.it
University of Bologna, Ravenna, Italy
Supplemental data for this article can be accessed online at https://doi.org/10.1080/10242422.2021.1913126
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
M. VAGNONI ET AL.
O
amines, carbohydrates and alcohols as hydrogen bond
donors (HBD). They are liquid at or below 100 ꢀC,
thanks to H-bond interactions between the single
components that create specific supramolecular struc-
tures. The number and the spatial positions of hydro-
gen atoms in the donor and acceptor groups,
available for hydrogen bonding, influence the forma-
tion and stability of the DES itself (Nkuku and LeSuer
2007; Zhang et al. 2012; Paiva et al. 2014; Smith et al.
O
O
H2O2
R
or
R’
R
OH
R
O
R’
CAL-B in
sugar NaDES
O
O
R
H2O
OH
or
R
O
R’
R
R’
Scheme 1. Chemoenzymatic pathway for epoxidations and
Baeyer-Villiger oxidations.
ꢀ
2014; Tommasi et al. 2017; Samorı et al. 2019). Dai
side reactions via oxirane ring opening, leading to
diols, hydroxyesters, and dimers. Prileshajev-epoxida-
tion can be chemoenzymatically carried out on various
substrates with CAL-B, a carboxylic acid as precursor
of the peracid, and H2O2 as oxidant (Scheme 1); this
method allows an improvement in terms of sustain-
ability, mild reaction conditions, limited side products
and use of less toxic reagents (Niku-Paavola and
Viikari 2000; Moreira and Nascimento 2007; Silva et al.
2011; Hollmann et al. 2011).
et al. (2013) reported numerous preparations of Natural
Deep Eutectic Solvents (NaDESs) by using plant metab-
olites. Interestingly, when water is added to the mix-
tures, in different proportions according to the NaDES,
it can be incorporated into this structure and becomes
strongly bound, reducing the viscosity of the NaDES
while retaining its original characteristics.
The chemoenzymatic oxidation systems described
above (Scheme 1) have been studied in several sol-
vents, including DESs (Gorke et al. 2008; Kotlewska
The B-V oxidation is a very well-known and useful
reaction for the synthesis of esters and lactones start-
ing from ketones, important building blocks in
pharmaceutical and polymer synthesis (Renz and
Meunier 1999; ten Brink et al. 2004; Woodruff and
Hutmacher 2010). Peracids such as meta-chloroper-
benzoic acid or peracetic acid are used as stoichiomet-
ric reagents, but also various catalytic methods that
use metals have been studied (Strukul 1998; Ma et al.
2014). Protocols based on B-V monooxygenases have
also been developed, but given their need for oxygen,
cofactor NADPH and their intrinsic low stability, they
are considered unpractical (Alphand et al. 2003; Leisch
et al. 2011; Balke et al. 2012). Nevertheless, the simple
chemoenzymatic method based on CAL-B, used for
epoxidation of alkenes described above, has been also
applied to this kind of oxidations (Scheme 1) (Lemoult
_ _
et al. 2011; Durand et al. 2012; Drozdz et al. 2015; Yang
and Duan 2016; Zhou et al. 2017; Ranganathan et al.
2017; Gotor-Fernandez and Paul 2019; Ma et al. 2019),
in which it has been demonstrated that the enzymatic
activity, stability, and selectivity can be enhanced (Zhou
et al. 2017; Ulger and Takac¸ 2017; Oh et al. 2019;
ꢁ
€
ꢁ
Guajardo et al. 2019; Gotor-Fernandez and Paul 2019;
Nian et al. 2020). Among the various NaDESs proposed
by Dai et al. we focused on the only one sugar-derived
and chlorine-free combination, composed by glucose,
fructose, sucrose and water (1:1:1:11), that we called
with the acronym GFS. To the best of our knowledge,
the lipase/H2O2 system was never reported in a solvent
like GFS and, following our interest on biocatalysis in
sustainable reaction media (Galletti et al. 2007), herein
we report on its application in the epoxidation of
alkenes and B-V oxidation of ketones.
ꢁ
et al. 1995; Rıos et al. 2007; Rios et al. 2008).
Green solvents exploited in chemoenzymatic oxida-
tion reactions can be categorised into two main
groups: (i) water and (ii) non-aqueous solvents like
ionic liquids (Moniruzzaman et al. 2010; Elgharbawy
et al. 2020), supercritical fluids and fluorinated solvents
(Hobbs and Thomas 2007). Despite their interesting
properties and application possibility, ionic liquids suf-
fer from several drawbacks like cost, toxicity, low bio-
degradability, complexity in preparation and handling
2. Materials and methods
2.1. Material
All chemicals and solvents were purchased from
Sigma-Aldrich or Alfa Aesar and used without any fur-
ther purification.
CAL-B (Lipase B from Candida antarctica) immobi-
lised on Immobead 150, recombinant from yeast,
4000 U/g was used.
ꢀ
(Samorı et al. 2015; Lei et al. 2017; Wang et al. 2017).
Deep Eutectic Solvents (DESs), described for the first
time by Abbott et al. (2001) are low melting mixtures
based on a combination of readily-available and inex-
pensive components, like quaternary ammonium salts
as hydrogen bond acceptors (HBA), and acids, amides,
2.2. DESs preparation
The components were mixed with the appropriate
stoichiometric ratios, heated at about 80-90 ꢀC (120 ꢀC

BIOCATALYSIS AND BIOTRANSFORMATION
3
Table 1. Epoxidation of cyclic alkenes with chemoenzymatic method in NaDES.
Peracid
precursor
(eq)
By-products
conversion
(%)a
CAL-B
(U/mmol)
Product
Entry
Alkene
NaDES
conversion (%)a
OH
OH
OH
Cl
2a’
34
2a”
59
1a
1a
2a
1
ChCl- Sorb [1:1]
250
OA, 1
6
-
1b
1b
2bb
2
ChCl- Sorb [1:1]
250
OA, 1
68
3
4
1b
1b
GFS
250
250
OA, 1
OA, 1
71
64
GFS [1:1:1] -LA
5
6
7
8
1b
1b
1b
1b
GFS [1:1:1] -LA
250
250
25
LA
15
75
GFS
GFS
GFS
OA, 0.1
OA, 1
80
25
OA, 0.1
79 (77)
-
1c
1c
2c
9
GFS
GFS
GFS
GFS
GFS
GFS
250
250
250
250
250
25
OA, 1
BA, 1
>99
10
11
12
13
14
1c
1c
1c
1c
1c
99
48
AA, 1
LA, 1
87
OA, 0.1
OA, 0.1
93
95 (91)
OH
OH
1d
2d
2d’
15c
GFS
60
OA, 1
1d
1d
76
73
14
16
16c
17c,d
18c,d
19c
GFS
GFS
GFS
GFS
60
60
30
30
OA, 0.1
OA, 1
1d
1d
1d
89
96
53
-
-
OA, 0.1
OA, 0.1
31
b
c
d
aconversion by GC-MS, isolated yield in brackets; diastereomeric ratio Z/E in 1b and 2b is always 2:1; Room temperature (20 ꢀC); H2O2 total amount
divided into 4 portions added in 4 hours.
ChCl: choline chloride; Sorb: sorbitol; GFS: glucose, fructose, sucrose, H2O (1:1:1:11); OA: octanoic acid; LA: levulinic acid; AA: acetic acid; BA: butyric acid.
for GFS) and magnetically stirred until homogeneous
liquid was obtained; for GFS, distilled water (up to
30 wt %) was then added to get a homogeneous col-
2.3. Representative procedure for enzymatic
epoxidations of alkenes
In a 4-mL vial, the immobilised CAL-B (amounts
reported in Tables 1–2 and Scheme 2) and 400 mg of
ourless liquid phase. All the DESs were cooled to rt
(20 ꢀC) before the use and stored in the fridge (4 ꢀC).

4
M. VAGNONI ET AL.
Table 2. Epoxidation of trans- stilbene 1e with chemoenzy-
matic method in GFS NaDES.
crudes were extracted with ethyl acetate and analysed
1
by GC-MS as described above. H and 13C NMR spec-
CAL-B
Peracid precursor
tra of some purified products have been acquired
after purification (see section 2.6) of the crude by
flash-column chromatography, some isolated yields
are also reported in Tables 3-4.
O
H O (1 eq.)
2
2
GFS
45 °C
48 h
1 e
2e
CAL-B
(U/mmol)
Peracid precursor
(eq)
Entry
2e conversion (%)a
1b
2b
3
250
250
250
25
OA, 1
OA, 0.1
OA, 1
OA, 1
DA, 1
HA, 1
BA, 1
AA, 1
60
traces
74 (70)
73
2.5. Silylation procedure
50 mL of silylating agent N,O-bis(trimethylsilyl)trifluor-
oacetamide and 1% chlorotrimethylsilane, (BSTFA þ
1% TMCS), 100 mL of CH3CN and 20 mL of pyridine
were added to 1–10 mg of sample into a GC-MS vial.
The vial was heated at 60–80 ꢀC for 30–40 min. The
sample was then diluted with CH3CN before
the injection.
4
5
25
11
6
25
54
7
25
-
8
25
-
aconversion by GC-MS, isolated yield in brackets; time (20 h).
GFS: glucose, fructose, sucrose, water (1:1:1:11); OA: octanoic acid; DA:
dodecanoic acid; HA: hexanoic Acid; BA: butyric acid; AA: acetic acid.
b
2.6. Purification procedure of selected products
DES (200 mg for 1d, 800 mg for 1e) were weighted,
followed by the addition of 1.6 mmol of alkene
(0.8 mmol for 1f), carboxylic acid (amounts reported in
Tables 1–2 and Scheme 2) and 1 equivalent (eq) of
H2O2 (30% aqueous solution). For entries 17 and 18 in
Table 1, H2O2 has been added in 4 aliquots in 4 h, for
substrate 1f 1.5 eq has been used.
The vial was heated at 45 ꢀC (or rt, 20 ꢀC, for 1d) for
various reaction times, then crudes were extracted
with cyclohexane or ethyl acetate and analysed by
GC-MS. Extraction residues were checked after deriva-
tization by silylation for the presence of other by-
products (see section 2.5 ). Conversions were calcu-
lated as ratios between products areas and total areas.
1H and 13C NMR spectra of some products have been
acquired after purification of the crude by flash-col-
umn chromatography (see section 2.6), some isolated
yields are also reported in the Tables. All products are
known, they were recognised by comparison with
standards or through mass spectra matching to what
reported in NIST database. Formation of by-products
was checked by GC-MS and NMR.
Reaction mixtures were extracted with ethyl acetate or
cyclohexane then washed with a NaHCO3 solution to
remove the octanoic acid (OA). After evaporating the
solvent, the crude was purified by flash chromatog-
raphy. The fractions containing the product were
mixed, the solvent evaporated, and the purified prod-
ucts were analysed by GC-MS and NMR (see spectra in
supplementary informations, SI).
2.7. Instrumentation
GC-MS analysis of epoxides and ester 4d were per-
formed using an Agilent HP 6850 gas chromatograph
connected to an Agilent HP 5975 quadrupole mass
spectrometer. Analytes were separated on a HP-5MS
fused-silica capillary column (stationary phase 5%-phe-
nyl)-methylpolysiloxane, 30 m, 0.25 mm i.d., 0.25 lm
film thickness), with helium as the carrier gas (at con-
stant pressure, 36 cm sꢁ1 linear velocity at 200 ꢀC).
Mass spectra were recorded under electron ionisation
(70 eV) at a frequency of 1 scan sꢁ1 within the
12 ꢁ 600 m/z range. The injection port temperature
was 250 ꢀC. The temperature of the column was kept
at 50 ꢀC for 5 min, then increased from 50 to 250 ꢀC at
10 ꢀC minꢁ1 and the final temperature of 250 ꢀC was
kept for 12 min.
GC-MS analysis of Baeyer-Villiger products (except
4d) were performed using an Agilent 7820 A gas chro-
matograph connected to an Agilent 5977E quadrupole
mass spectrometer. Analytes were separated on a DB-
FFAP polar column (30 m length, 0.25 mm i.d., 0.25 mm
film thickness), with helium flow of 1 mL minꢁ1. Mass
spectra were recorded under electron ionisation
2.4. Representative procedure for B-V oxidation
of ketones
In a 4-mL vial, the immobilised CAL-B (amounts in
Tables 3-4) and 400 mg of GFS for 3a, 3 b, 3c, (700 mg
of GFS for 3d) were weighted, followed by the add-
ition of 0.8 mmol of ketone (0.4 mmol for 3d), carbox-
ylic acid (amounts in Tables 3-4) and 1 eq of H2O2
(30% aqueous solution), different amounts of H2O2 are
reported in Tables 3-4. The vial was heated at 45 ꢀC or
at kept at rt, 20 ꢀC, for various reaction times, then

BIOCATALYSIS AND BIOTRANSFORMATION
5
Table 3. Baeyer-Villiger oxidation of lactones with chemoenzymatic method in GFS.
CAL-B
Octanoic Acid
H2O2
O
O
R2
R1 R2
GFS
T°C
time
R1
O
3a-c
4a-c
By-product
conversion
(%)la
CAL-B
(U/mmol)
OA
(eq)
H2O2
(eq)
T
(°C)
time
(h)
Product
Entry
Ketone
conversion (%)a
4a’
3a
3a
3a
4a
32
23
21
20
70
20
-
53
16
1
2
125
250
1
1
1
1
20
20
5
15
35
36
-
28
3
3a
200
1
1
45
20
40
20
40
5
20
20
40
64
5
20
20
4d
20
4d
15
15
37
55
30
20
49
61
74
52
29
36
54
58
54
-
-
-
-
-
8
-
-
-
-
21
-
16
-
4
5
6
3a
3a
3a
125
125
125
0,1
1
1
2
2
20
20
45
1
7
3a
125
2
2
20
8
9
3a
3a
3a
125
125
125
2
1
3
2
3
3
45
20
20
10
16
3b
3b
4b
4b’
20
40
15
7
-
50
11
65
1
1
20
12
13
3b
3b
65
65
1
1
1
45
20
20
40
10
4
30
15
0.1
Not found
-
4c
3c
3c
14
65
various various
20
various
traces
aconversion by GC-MS, isolated yield in brackets.
GFS: glucose, fructose, sucrose, H2O (1:1:1:11); OA: octanoic acid.
(70 eV) at a frequency of 1 scan sꢁ1 within the
29 ꢁ 450 m/z range. The injection port temperature
was 250 ꢀC.
trimethylsilane (TMS) with the solvent resonance as
the internal standard (deuterochloroform: 7.26 ppm).
The temperature of the column was kept at 50 ꢀC fꢁo1r
5 min, then increased from 50 to 250 ꢀC at 10 ꢀC min
and the final temperature of 250 ꢀC was kept for 15 min.
1H NMR spectra were recorded on Varian 400
(400 MHz) spectrometers. 13C NMR spectra were
recorded on a Varian 400 (100 MHz) spectrometers.
Chemical shifts were reported in ppm from
3. Results and discussion
3.1. Alkenes epoxidation
We studied the chemoenzymatic epoxidation of vari-
ous alkenes (focusing for each of them on the enzyme
amount, peracid precursors and H2O2 amount and

6
M. VAGNONI ET AL.
additions, and reaction time): cyclic alkenes (Table 1),
poorly-reactive stilbene (Table 2) and oleic acid
(Scheme 2).
We tested both solvents (ChCl-Sorb and GFS) in the
same conditions with more easily detectable cyclodo-
cedene 1b and we observed that GFS gave better
conversions (Table 1, entries 2 and 3); the same held
true for other cyclic substrates (cyclooctene 1c and
limonene 1d) (see SI Table S1, entries 1 and 5, and
Table S2, entry 1). So, we decided to test various sub-
strates to check the viability of the system.
When Z/E mixtures were used in the starting alkene
(as in 1b), no diastereoselectivity was observed and
the final product diastereomeric ratio reflected the
diastereomeric distribution in the reagent.
OA resulted the most reactive acid precursor under
our conditions (Table 1, entries 9-12), confirming the
literature results, and its amount can be significantly
lowered from 1 eq to 0.1 eq with all the substrates
(Table 1, entries 6, 8, 13, 14, 18). Considering aliphatic
acids with different chain lengths, butyric acid BA
(Table 1, entry 10) gave very good results on 1c while
acetic acid AA (Table 1, entry 11) was poorly reactive;
the biobased levulinic acid LA gave 2c in good con-
version (Table 1, entry 12), prompting us to include
40% of LA as a component of the GFS instead of
water, with the aim of using it both as peracid precur-
sor and solvent component; however in this case the
epoxidation of cyclododecene 1b was not satisfactory
(Table 1, entry 5). We also tested GFS-LA in combin-
ation with OA as peracid precursor on 1 b; results
were good but lower than using GFS (Table 1, entries
3 and 4). The same happened with 1c (see SI, Table
S1, entry 4). Dimethyl carbonate DMC was also tested
as peracid precursor but without good results (see SI,
Table S1, entries 1 and 3). The amount of the enzyme
could be lowered till 30-25 U/mmol without significant
loss of reactivity (Table 1, 1b entries 7 and 8, 1c entry
14, 1d entries 18 and 19).
3.1.1. Cyclic alkenes 1 a-d
Studying CAL-B mediated chemoenzymatic epoxida-
tion in various NaDESs, Zhou et al. showed that
amine-based DESs (i.e., choline chloride-urea, 1:2 molar
ratio, called Reline) significantly reduced the stability
of CAL-B in a wide temperature range whereas the
polyol-based ones increased it (Zhou et al. 2017). For
this reason, we focused our initial experiments on pol-
yol-based NaDESs. Cyclohexene 1a (Table 1, entry 1)
was epoxidized with immobilised CAL-B (100 mg per
1.6 mmol of alkene), octanoic acid OA (one eq respect
to the alkene), and H2O2 (one eq respect to the
alkene) in choline chloride-sorbitol (ChCl-Sorb), 1:1
molar ratio (400 mg) (similarly to Zhou et al). We
observed a complete conversion of the starting mater-
ial but a very low selectivity towards the epoxide; in
fact the most of epoxide was converted into the
chlorinated by-product and the diol after 20 h. This
unexpected result prompted us to turn our attention
towards chloride-free, sugar-based NaDESs as the GFS.
Table 4. Baeyer-Villiger oxidation of 2-phenylacetophenone 3d
in GFS.
CAL-B
(U/mmol)
OA
(eq)
H2O2
(eq)
time
(h)
40
Conversion 4d
Entry Ketone
(%)a,c
7
1b
3d
250
1
1
50
17
7 days
50
2b
3
3d
3d
250
250
0.1
1
1
1
50
12
40
7 days
20
5 days
20
50
3 days
23
4 days
40 (34)
50
18
27
38
42
44
55
60
4
5
6
3d
3d
3d
100
250
250
1
2
1
1
2
3
R-limonene 1d is a very important biobased sub-
strate, whose epoxy derivative has some relevance in
the field of polymer synthesis (Auriemma et al. 2015).
Its internal double bond is much more reactive than
the terminal one, being electron-richer; in all the
tested conditions the product 2d was obtained. Since
we initially observed the formation of the diol as by-
product (Table 1, entry 15), milder conditions were
b
c
aconversion by GC-MS, isolated yield in brackets; temperature (20 ꢀC); in
all the entries there are traces of the regiosomer of 4d,
O
4d’
.
O
GFS: glucose, fructose, sucrose, H2O (1:1:1:11); OA: octanoic acid.
CAL-B 120 U/mmol
O
O
H2O2 1.5 eq.
OH
OH
6
6
6
6
GFS 400 mg
45 °C
O
20h
1f
2f
GC-MS Yield: 95%
Isolated Yield: 85%
Scheme 2. Epoxidation of oleic acid with chemoenzymatic method in GFS.

BIOCATALYSIS AND BIOTRANSFORMATION
7
tested: i) halving the amount of NaDES; ii) keeping the
temperature below 25 ꢀC; iii) lowering addition rate of
H2O2. All these conditions avoided the diol formation
(Table 1, entries 17 and 18). The use of a catalytic
amount of acid precursor (Table 1, entry 16) and a
lower amount of the enzyme defined the best condi-
tions to obtain 2d in very good conversion (Table 1,
entry 18).
epoxidation can also be carried out in a solvent-free
system, but the process is more efficient for methyl
oleate since the corresponding epoxide is liquid
respect to solid 9,10-epoxystearic acid (Orellana-Coca
et al. 2005).
3.2. Baeyer-Villiger oxidations
The first use of CAL-B as catalyst for B-V oxidations
was performed in toluene with myristic acid as peracid
precursor (Lemoult et al. 1995). Recent examples
report ethyl acetate both as solvent and peracid pre-
cursor (therefore in large excess with respect to the
As expected from limonene results, terminal bonds
of styrene and itaconic anhydride were not reactive in
the mild condition we tested for the other substrates
(see SI, Table S2, entries 5 and 6).
ꢁ
starting material) (Rıos et al. 2007; Rios et al. 2008;
3.1.2. Trans-Stilbene 1e
ꢁ
_ _
Chavez et al. 2013; Drozdz et al. 2013) and combin-
ation of ionic liquids and OA as solvent and peracid
precursor, respectively (OA in excess with respect to
trans-Stilbene 1e is a challenging substrate because its
double bond is electron-poor and it is poorly soluble
in polar solvents like GFS. OA and other linear ali-
phatic carboxylic acids with shorter (hexanoic HA,
butyric BA and acetic acid AA) and longer (dodecanoic
acid, DA) chain lengths were tested as peracid precur-
sors (Table 2). In all cases OA resulted the most effect-
ive acid precursor, but 1 eq was needed to obtain an
effective conversion (Table 2, entry 2). A decrease of
the enzyme amount was possible, but a conversion of
75% was reached only after 48 h (Table 2, entry 4).
Longer reaction times did not increase the conversion
(see SI, Table S2, entries 4). Differently, the electron-
poor, a-b double bonds of crotonic acid and methyl
crotonate were very difficult to be epoxidized (see SI,
Table S2, entries 7 and 8) and we obtained just traces
of the products. We also tested substrates carrying
hydroxyl groups such as 1-octen-3-ol or trans-2-hexen-
1-ol but, as expected, the main product was the ester
formed by OA and the alcohol under CAL-B catalysis
(data not shown).
_ _
the starting ketone) (Kotlewska et al. 2011; Drozdz
et al. 2015). Urea-hydrogen peroxide is considered a
milder oxidant than H2O2 alone and it was used to
reduce the formation of water in the reaction (Rıos
et al. 2007; Rios et al. 2008), nevertheless other studies
showed no significant improvement in product con-
ꢁ
ꢁ
version and enzyme recycling (Chavez et al. 2013).
Considering that water is already present in our GFS,
the availability and the lower cost of hydrogen perox-
ide, this last one was thus chosen as oxidant in our
study. As for reaction times, when the reaction was
carried out at room temperature it generally required
very long reaction times (in the order of days) to reach
ꢁ
effective conversions (Rıos et al. 2007; Rios et al. 2008;
ꢁ
_ _
_ _
Chavez et al. 2013; Drozdz et al. 2013; Drozdz
et al. 2015).
We tested B-V oxidation on various substrates (see
section 2.4), using the same chemoenzymatic method
in GFS previously described for epoxidation reactions:
CAL-B, H2O2 (30% aqueous solution) and OA as pera-
cid precursor (scheme in Table 3). Also in this case, a
detailed study was conducted on the reaction condi-
tions, with the aim of reducing the use of the reagents
in excess and to use the mildest possible conditions.
3.1.3. Oleic acid 1f
Oleic acid 1f is a very interesting substrate since its
epoxide (9,10-epoxystearic acid) is a highly-valuable
oleochemical due to its wide range of industrial appli-
cations, including cosmetics, personal care, and
pharmaceutical products. The epoxidation worked
very well and without the addition of OA (Scheme 2),
thanks to an autocatalytic mechanism that formed the
3.2.1. Cyclic ketones 3a-c
Since highly reactive in B-V oxidations, cyclohexanone
3a was the first substrate tested. By carrying out the
reaction at 20 ꢀC, lactone 4a (e-caprolactone, Table 3,
entry 1) was obtained but the reaction proceeded
very slowly and an increase in time led to the forma-
tion of the by-product, 6-hydroxyhexanoic acid 4a’,
caused by the ring-opening of 4a. Increasing the
amount of catalyst or temperature did not increase
the selectivity towards 4a formation (Table 3, entries 2
€
peroxy acid from the oleic acid itself (Rusch Gen. Klaas
and Warwel 1999). A temperature of 45 ꢀC was
required not only to catalyse the reaction but also to
avoid the product solidification. The conditions used
are the same reported by the recent literature (tem-
perature at maximun 50 ꢀC, an excess of H2O2, short
reaction time), except for the use of the solvent, which
is generally toluene (Milchert et al. 2015). The

8
M. VAGNONI ET AL.
and 3). Differently from the epoxidation reaction of
cyclic alkenes (Table 1), the use of the peracid precur-
sor in catalytic amount did not give good results
(Table 3, entry 4). Indeed, x-hydroxy acid formation is
the main drawback in CAL-B mediated B-V oxidation
(amounts reported in Table 3) (Wang et al. 2017).
Increasing the amount of H2O2 to 2 eq (Table 3,
entries 5 and 6) gave higher conversions, without the
formation of any by-product, while a shorter reaction
time was achieved by conducting the reaction at 45 ꢀC
(Table 3, entry 6). The use of both OA and H2O2 in
excess (Table 3, entries 7 and 8) gave the best conver-
sion: 74% at 20 ꢀC and 52% at 45 ꢀC. As previous
observed when the reaction was conducted at 45 ꢀC,
the reaction must be stopped after a few hours to
avoid by-product formation (Table 3, entry 8). Further
increasing of both oxidant and acid amounts was not
effective (Table 3, entry 9 and 10).
mixture of glucose, fructose, sucrose and water (GFS).
Specific conditions to perform the reaction on selected
substrates in good conversion and selectivity were
found. The best conditions for epoxidations proved to
be related to the substrate reactivity; reaction condi-
tions were tuned and catalysts amounts decreased to
obtain epoxides from poorly reactive and steric-hin-
dered double bonds (as trans-stilbene) and to control
the formation of byproducts in more reactive alkenes
(like internal double bond of R-limonene). Baeyer-
Villiger oxidations always required at least stoichiomet-
ric amount of the peracid precursor to proceed and
an excess of both oxidant and acid to obtain good
conversions.
Acknowledgements
The authors thank the EMM ChIR (Chemical innovation and
regulation) for M.K. Vasquez De Paz and D.G. Gidey thesis
fellowship, the University of Bologna (RFO program) and
MIUR (PhD program) for funding.
The expected higher reactivity of cyclopentanone
3b prompted us to lower the enzyme amount but
also tuning temperature, oxidant and OA amounts
(Table 3, entries 11-13) the high reactivity of the sub-
strate caused a rapid formation of the by-product 3b’.
Disclosure statement
ꢁ
As expected from the literature (Chavez et al. 2013;
No potential conflict of interest was reported by
the author(s).
_ _
_ _
Drozdz et al. 2013; Drozdz et al. 2015), substrates with
larger rings, as cyclooctanone 3c, are unreactive in all
the tested conditions (Table 3, entry 14).
ORCID
Martina Vagnoni
http://orcid.org/0000-0001-6812-6951
3.2.2 2-Phenylacetophenone 3d. When using 2-phe-
nylacetophenone 3d the regioselectivity issue must be
considered, due to the formation of two possible
regioisomers 4d and 4d’ (structure in Table 4 foot)
caused by the migration of the phenyl group instead
of the benzyl one (favored). As expected, we predom-
inantly obtained regioisomer 4d in 50% conversion at
long reaction times (Table 4, entry 1). Higher tempera-
ture did not increase the conversion but significantly
increased the reaction rate (Table 4, entries 1 and 3).
Conversions decreased by lowering the amount of OA
and enzyme (Table 4, entries 2 and 4). Using 2 eq of
OA and H2O2 was not effective (Table 4, entry 5),
while a great excess of H2O2 gave 60% of 4d (Table 4,
entry 6). Linear ketones and levulinic acid were tested
but the reaction did not work under the developed
conditions (data not shown).
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