Chemoenzymatic epoxidation of alkenes based on peracid formation by a Rhizomucor miehei lipase-catalyzed perhydrolysis reaction

Article abstract of DOI:10.1016/j.tet.2013.12.084

A chemoenzymatic and selective method for the epoxidation of a series of cyclic and linear alkenes is described. Epoxides have been obtained in moderate to excellent conversions under mild reaction conditions through a two-step sequence, carried out in one-pot. This chemoenzymatic approach is based on a Rhizomucor miehei lipase-catalyzed perhydrolysis reaction to form the corresponding peracid, and subsequent epoxidation of the corresponding alkenes. Reaction parameters with influence in the biotransformation have been optimized specially focusing in the efficient enzymatic peracid formation by means of the correct choice of solvent, oxidant, and peracid precursor. This chemoenzymatic approach has been efficiently applied for the first time, in the regioselective chemical oxidation of (S)-carvone and limonene, both showing an opposite behavior for the oxidation of the internal and external C-C double bond, respectively.

Full text of DOI:10.1016/j.tet.2013.12.084

Accepted Manuscript
Chemoenzymatic epoxidation of alkenes based on peracid formation by a
Rhizomucor miehei lipase-catalyzed perhydrolysis reaction
Daniel Méndez-Sánchez, Nicolás Ríos-Lombardía, Vicente Gotor, Vicente Gotor-
Fernández
PII:
S0040-4020(13)01952-2
10.1016/j.tet.2013.12.084
TET 25168
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 22 November 2013
Revised Date: 18 December 2013
Accepted Date: 26 December 2013
Please cite this article as: Méndez-Sánchez D, Ríos-Lombardía N, Gotor V, Gotor-Fernández V,
Chemoenzymatic epoxidation of alkenes based on peracid formation by a Rhizomucor miehei lipase-
catalyzed perhydrolysis reaction, Tetrahedron (2014), doi: 10.1016/j.tet.2013.12.084.
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ACCEPTED MANUSCRIPT
Graphical Abstract.
Chemoenzymatic epoxidation of alkenes based on
peracid formation by a Rhizomucor miehei lipase-
catalyzed perhydrolysis reaction
Leave this area blank for abstract info.
Daniel Méndez-Sánchez, Nicolás Ríos-Lombardía, Vicente Gotor, and Vicente Gotor-Fernández*
Departamento de Química Orgánica e Inorgánica, Universidad de Oviedo, Instituto Universitario de
Biotecnología de Asturias, c/ Julián Clavería 8, 33006 Oviedo, Spain. E-mail address: vicgotfer@uniovi.es
R2
R2
MeCN, 24 h, 30 ºC
O
R3
R3
R1
R1
H
H
O
O
18 to >97% yield
OH
12 examples
R4
OOH
R4
H2O
"H2O2"
Rhizomucor miehei lipase

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Chemoenzymatic epoxidation of alkenes based on peracid formation by a
Rhizomucor miehei lipase-catalyzed perhydrolysis reaction
Daniel Méndez-Sánchez, Nicolás Ríos-Lombardía, Vicente Gotor, and Vicente Gotor-Fernández*
Departamento de Química Orgánica e Inorgánica, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, c/ Julián Clavería 8, 33006
Oviedo, Spain.. Telephone: +34 985 103454. Fax number: +34 985 103446; E-mail address: vicgotfer@uniovi.es
ARTICLE INFO
ABSTRACT
Article history:
Received
A chemoenzymatic and selective method for the epoxidation of a series of cyclic and lineal
alkenes is described. Epoxides have been obtained in moderate to excellent conversions under
Received in revised form
Accepted
Available online
mild reaction conditions through a two-step sequence, carried out in one-pot. This
chemoenzymatic approach is based on a Rhizomucor miehei lipase-catalyzed perhydrolysis
reaction to form the corresponding peracid, and subsequent epoxidation of the corresponding
alkenes. Reaction parameters with influence in the biotransformation have been optimized
specially focusing in the efficient enzymatic peracid formation by means of the correct choice of
solvent, oxidant and peracid precursor. This chemoenzymatic approach has been efficiently
applied for the first time, in the regioselective chemical oxidation of (S)-carvone and limonene,
both showing an opposite behavior for the oxidation of the internal and external C-C double
bond, respectively.
Keywords:
Keyword_1: Alkenes
Keyword_2: Biocatalysis
Keyword_3: Epoxidation
Keyword_4: Hydrolases
Keyword_5: Rhizomucor miehei lipase
2009 Elsevier Ltd. All rights reserved.
1. Introduction
to the peracid, which is the responsible for the chemical
oxidation of the alkene. In this manner, peracid storage and
Epoxides represent an important class of oxygenated
heterocycles with multiple applications in the fine chemical
industry for the production of adhesives, coating, paints and
polymers. Additionally, they are considered as versatile synthetic
intermediates towards alcohols, alkenes, diols and other families
of interesting organic compounds. Traditionally epoxides have
been smoothly prepared through heterogeneous or homogeneous
catalytic alkene epoxidation, using metal catalysts, organic
peroxides or peracids for non enzymatic transformations,1 while
monooxygenases or chlorperoxidases have been employed in
biotransformations. Remarkably, the asymmetric version has
received great attention in the last decades for the production of
the desired epoxides generally with excellent yields and
enantiomeric excess.2
transportation is not required, providing a safe strategy for the
development of different oxidative transformations,5 such as
consecutive epoxidation and esterification reactions,6 Baeyer
Villiger oxidation of cyclic ketones7 or lignocelluloses
delignification.8 Interestingly, some recent studies of this
biocatalytic promiscuity behavior display by hydrolases9 have
concluded that the oxidation step is strictly a chemical step that
occurs without the action of the enzyme, and for that reason non
stereoselectivity is achieved.3c In fact, moderate to good
stereoselectivities have been obtained when using chiral oxygen
transferring agents.10
R2
R2
O
R4
R4
R1
R1
R3
R3
One of the most common strategies for the synthesis of
epoxides is based on in situ peracid formation (perhydrolysis),
which can be achieved by reaction of a carboxylic acid or ester
with hydrogen peroxide or their derivatives as oxidants. From an
environmental point of view, hydrogen peroxide is a preferred
oxidant since the transformation gives only water as by-product.
In this context, the enzymatic-catalyzed epoxidation of alkenes
has been successfully achieved using hydrolases such as
perhydrolases, lipases, esterases and acyl transferases.3 For this
type of transformation, also called Prileshajev epoxidation,
lipases are usually the biocatalyst of choice, especially since
Bjorkling and co-workers described their applicability for the
first time in 1990 (Scheme 1).4 This is a very practical reaction as
combines in one-pot, the oxidation of the carboxylic acid or ester
O
O
R5
OOH
Lipase
R5
OH
H2O
H2O2
Scheme 1. Epoxidation of alkenes through lipase-catalyzed
perhydrolysis of carboxylic acids.
The lipase-catalyzed approach can be considered as an
indirect epoxidation reaction of alkenes, although some authors
have demonstrated the potential of Candida antractica lipase
type B (CAL-B) mutants for catalyzing the direct epoxidation of
α,β-unsaturated aldehydes with hydrogen peroxide.11 CAL-B12

2
Tetrahedron
and Pseudomonas cepacia lipase (PSL)13 are probably the most
Table 1. Epoxidation of cyclohexene (1a, 0.33 M) using
ACCEPTED MANUSCRIPT
versatile lipases for alkene epoxidation using a variety of peracid
precursors. However little attention has been made to other
RML, carboxylic acids or EtOAc (1.1 eq) and UHP (1.1 eq) in
different organic solvents at 30 ºC and 250 rpm after 5 h.
hydrolases.4,14 Rhizomucor miehei lipase (RML) is
a
UHP, R1COOR2
commercially available enzyme in both soluble and immobilized
form, which has shown excellent activities in a series of synthetic
RML
O
Organic solvent
5 h, 30 ºC, 250 rpm
applications
such
as
aminolysis,
esterification
or
transesterification,15 however its application in perhydrolysis
reactions for the later formation of alkenes remains in a
premature stage as negligible4,14 or moderate16 conversions were
found by different authors, probably because of the inactivation
of the enzyme at high hydrogen peroxide concentrations.
1a
2a
Entry
R1COOR2
Solvent
EtOAc
THF
c (%)a
5
1
EtOAc
2
Decanoic acid
Decanoic acid
Decanoic acid
Decanoic acid
Acetic acid
4
Herein, we wish to report the application of RML in the
synthesis of a broad panel of epoxides, dealing with the
optimization of several parameters with influence in the enzyme
reactivity. Thus, the production of twelve epoxides in good
overall yields through a two-step and one-pot process will be
discussed, two of them in a selective fashion were the oxidation
of the internal or external carbon-carbon double bond was
achieved.
3
2-Me-THF
TBME
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
16
16
23
<3
<3
31
20
<3
<3
4
5
6
7
Butanoic acid
Lauric acid
8
9
Stearic acid
Benzoic acid
Phenylacetic acid
2. Results and discussion
10
11
The initial objective was to find the optimal conditions for the
peracid formation. Cyclohexene (1a) was considered as a model
substrate. because of its commercial availability and easiness in
the monitoring of its epoxidation reaction by gas chromatography
(GC). Different oxidants were used to carry out the oxidation
reaction towards the formation of cyclohexene oxide (2a).
Initially 4 equivalents of a 30% hydrogen peroxide aqueous
solution were used in the presence of decanoic acid with a water:
dichloromethane mixture (1:1.2 v/v) as solvent. Unfortunately
deactivation of RML was rapidly observed, yielding 2a in only
4% conversion after 24 h at 30 ºC. This result is far to be
synthetically applicable in comparison with the complete
conversion achieved with CAL-B as catalyst. The use of
dimethyl carbonate12c as peracid precursor also led to negligible
conversions. Then, hydrogen peroxide-urea complex (UHP) was
used for the safe release of hydrogen peroxide into the medium,
and a first screening was conducted trying to avoid the presence
of great amounts of H2O2 that clearly deactivate RML (Table 1).
a Conversion values determined by GC.
The effects of temperature and alkene concentration were later
analyzed in depth trying to improve the conversion values
reached with lauric acid for the epoxidation of cyclohexene. For
that reason the best conditions previously found were used for the
epoxidation of 1a, which means 1.1 equivalents of lauric acid and
UHP in acetonitrile (Table 2). The conversions found for the
reactions at 37 ºC and 45 ºC were significantly lower in
comparison with the transformation at 30 ºC (entries 1-3), fact
probably related to the thermostability of RML. Remarkably,
maintaining the temperature at 30 ºC but doubling the substrate
concentration a 64% conversion into 2a was reached after 5 h
(entry 4), and 85% after 24 h (entries 5 and 6). However a
decrease of reactivity was observed at higher concentrations (1 M
of 1a, entry 7).
Table 2. Epoxidation of cyclohexene (1a) using RML, lauric
acid (1.1 eq) and UHP (1.1 eq) in MeCN at different
concentrations and 250 rpm.
First, ethyl acetate was used as both solvent and carboxylic
acid derivative to form the peracid, however only 5% conversion
was achieved after a short time span of 5 h (entry 1). Then,
different solvents were used in combination with decanoic acid
and UHP, while tetrahydrofuran led to a very low conversion
(entry 2), both 2-methyltetrahydrofuran and tert-butymethyl ether
led to 16% (entries 3 and 4), the highest conversion being
achieved with acetonitrile (entry 5). It must be mentioned that the
reaction in the absence of enzyme led to negligible conversions
in all cases.
UHP, Lauric acid
RML
O
MeCN
5 h, 250 rpm
1a
T (ºC)
2a
t (h)
Entry
[1a] (M)
0.33
0.33
0.33
0.66
0.66
0.66
1
c (%)a
31
1
2
3
4
5
6
7
30
37
45
30
30
30
30
5
5
12
The nature of carboxylic acid was then studied, finding that
short-chain carboxylic acids (i.e. acetic acid or butyric acid,
entries 6 and 7) did not serve as good intermediates for the
peracid formation. The best result was found for lauric acid (31%
conversion), containing two carbon atoms more than decanoic
acid (entries 5 and 8), while a decrease in the conversion was
found using a bigger one such as stearic acid (entry 9). In
addition, no conversion was found for those containing aromatic
rests in their structure (entries 10 and 11).
5
9
5
64
13
24
5
75
85
43
a Conversion values determined by GC.
Next, we decided to analyze the influence of both the UHP
and lauric acid concentrations using different loadings of RML.
The results have been summarized in Table 3. First, a clear

3
enzyme inhibition was observed at higher concentration of the
oxidant UHP (entries 1 and 2), which is not surprising as that was
previously observed when using the own hydrogen peroxide as
oxidant. For that reason we decided to focus on different
concentrations of lauric acid (0.2-1.1 eq.), observing that the
reaction evolve efficiently towards the formation of the epoxide
2a at higher concentrations of lauric acid (entries 3-8). In order,
to reach better results the reaction was left for 24 h at both 1.1
and 2 equivalents of lauric acid, yielding 85 and 91%
conversions, respectively (entries 9 and 10). However a 3-fold
excess of lauric acid led to a poor solubility of the mixture in the
reaction medium, leading to a significant lower conversion (68%,
entry 11). Finally, it was demonstrated that the use of higher
loadings of RML did not lead to an improvement in the
conversion value after 24 h (entries 12 and 13).
was performed with 10 mmol of 1,4-dihydronaphtalene (1d),
ACCEPTED MANUSCRIPT
the reaction with magnetic stirring led to the same 82%
conversion value, finding a slight decrease with orbital shaking
conditions (62%).
One of the advantages when using immobilized enzymes, is
the possibility to recover the enzyme at the end of the process for
further applications. Enzyme recycling is a key issue in
biotransformations
especially
for
economic
reasons.
Unfortunately in the oxidation of 1,4-dihydronaphtalene (1d) the
reuse of the RML led to a premature and dramatic decrease in
activity. More than five-times lower activity was observed after
two cycles and a complete loss of activity in the third one. These
results suggest the denaturalization of the enzyme with a
prolonged exposure to hydrogen peroxide.
Table 3. Epoxidation of cyclohexene (1a) using RML, lauric
acid and UHP in MeCN at 30 ºC and 250 rpm.
Table 4. Epoxidation of alkenes 1a-j (0.66 M) using RML (50
mg/mmol), lauric acid (2 eq) and UHP (1.1 eq) in MeCN at 30 ºC
and 250 rpm after 24 h.
RML
Lauric acid
Lauric acid
H
H
UHP, lipase
O
MeCN
UHP, RML
O
H
H
R1
R1
MeCN
24 h, 30 ºC
250 rpm
30 ºC, 250 rpm
R2
1a-j
R2
1a
2a
2a-j
Entry
RML
UHP
(eq)
Lauric acid
(eq)
[1a] (M) t (h) c (%)b
(mg)a
50
Entry
Substrate
2a-j (%)a
91
1
1.1
2
1.1
1.1
0.2
0.4
0.6
0.8
1.0
1.1
1.1
2
0.33
0.33
0.66
0.66
0.66
0.66
0.66
0.66
0.66
0.66
0.66
0.66
0.66
5
31
12
3
1
Cyclohexene (1a)
2
50
5
2
2-Methyl-cyclohexene (1b)
Cycloheptene (1c)
>97
>97
82
3
50
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
5
3
4
50
5
13
24
38
51
64
85
91
68
80
85
4
1,4-Dihydronaphtalene (1d)
1,2-Dihydronaphtalene (1e)
4-Octene (1f)
5
50
5
5
83
6
50
5
6
80
7
50
5
7
Styrene (1g)
54
8
50
5
8
2-Bromo-Styrene (1h)
3-Bromo-Styrene (1i)
4-Bromo-Styrene (1j)
25
9
50
24
24
24
14
24
9
27
10
11
12
13
50
10
40
50
3
a
Percentage of epoxide 2a-j obtained from the
corresponding alkene. Determined by GC.
100
100
1.1
1.1
a Amount of RML in mgs for mmol of cyclohexene (1a).
b Conversion values determined by GC.
The oxidation of a range of epoxides is shown in Table 4,
using the optimum conditions for the epoxidation of cyclohexene
with RML, lauric acid and UHP. Gratifingly, the oxidation of
cyclic substrates such as 2-methylcyclohexene (1b) and
cyclohepetene (1c) occurred in quantitative yield after 24 h
(entries 2 and 3), while dihydronaphtalene derivatives 2d,e were
obtained as major products with conversion up to 82% (entries 4
and 5), observing the formation of diol in 16% from 1e by
epoxidation and subsequent hydrolytic opening of the oxirane. A
similar result was found for a linear alkene such as 4-octene (1f),
obtaining the epoxide 2f as major product in 80% (entry 6). Next,
we decided to explore the potential of our oxidative system using
styrene derivatives, observing a 54% conversion for the non
substituted one (1g, entry 7), while a low reactivity was observed
for styrenes 1h-j bearing a bromo substituent in the aromatic
ring. In these cases, the formation of epoxide was slighlty higher
for the para-substituted (entries 8-10). Remarkably, similar
results were achieved using magnetic stirring or orbital shaking
in a 1 mmol scale. However, when the scale-up of the reaction
Figure 1. Study of the RML recycling using 1d, lauric acid (2
eq) and UHP (1.1 eq) at 30 ºC during 24 h at 250 rpm.
Finally, we decided to explore the possibility to carry out
regioselective oxidation processes, which can have potential
applications in different areas such as the oxidation of terpenes.
In fact, just a few examples have been published in this area
related to the use of lipases for the indirect oxidation of C-C
double bonds.17 We selected compounds (S)-carvone and

4
Tetrahedron
ACCEPTED MANUSCRIPT
limonene both having two C=C bonds in their structure, one
Gas chromatography analyses were carried out in a HP6890
inserted in a cyclic ring and other exocyclic (Table 5).
Significantly limonene was oxidized using RML for the peracid
formation, affording with 18% conversion the epoxide derived
from the internal alkene bond oxidation as unique product (entry
1), while satisfyingly the oxidation of the exocyclic C=C bond of
chromatograph with FID detection. Two non chiral columns were
used for the identification and quantification of alkenes and
epoxides.
A Hewlett Packard HP5 (30 m x 0.32 mm x 0.25 µm) using
the following program 30 (°C)/ 3 (min)/ 15 (°C/min)/ 200 (°C)/ 2
(min), was used for 1a (2.8 min), 2a (5.4 min), 1b (4.4 min) and
2b (6.1 min).
(S)-carvone was achieved with
a more significant 78%
conversion (entry 2). The epoxidation reactions of limonene and
(S)-carvone using CAL-B were also run for comparison, finding
similar results in terms of activity (entries 3 and 4)
A Hewlett Packard HP1 (30 m x 0.32 mm x 0.25 µm) with the
following programs of temperatures:
Table 5. Epoxidation of limonene and (S)-carvone (0.66 M)
using RML (50 mg/mmol), lauric acid (2 eq) and UHP (1.1
eq) in MeCN at 30 ºC after 24 h at 250 rpm.
UHP
a) 50 (°C)/ 3 (min)/ 15 (°C/min)/ 200 (°C)/ 3 (min) for 1c (2.1
min), 2c (4.4 min), 1f (2.2 min) and 2f (4.3 min).
b) 90 (°C)/ 3 (min)/ 15 (°C/min)/ 200 (°C)/ 3 (min) for 1d (4.0
min), 2d (6.4 min), 1e (8.1 min), 2e (10.0 min), 1h (3.6 min), 2h
(5.3 min), 1i (3.8 min), 2i (5.8 min), 1j (7.0 min) and 2j (8.9
min).
O
Y
CH3(CH2)10COOH
RML
O
X
or
MeCN
24 h, 30 ºC
250 rpm
O
c) 70 (°C)/ 3 (min)/ 15 (°C/min)/ 200 (°C)/ 3 (min) for 1g (2.0
min) and 2g (4.1 min).
1k (Y-C-X = CH2)
1l (Y-C-X = C=O)
2k
2l
d) 120 (°C)/ 3 (min)/ 15 (°C/min)/ 200 (°C)/ 3 (min) for 1k (1.9
min), 2k (3.0 min), 1l (3.7 min) and 2l (5.7 min).
Entry
Substrate
Enzyme
c (%)a
1
2
3
4
Limonene (1k)
(S)-Carvone (1l)
Limonene (1k)
(S)-Carvone (1l)
RML
18 (2k)
78 (2l)
20 (2k)
80 (2l)
4.3. General procedure for the epoxidation of alkenes at 1 mmol
scale.
RML
CAL-B
CAL-B
Lauric acid (2 mol, 400 mg), RML (50 mg) and finally UHP (1.1
mmol, 104 mg) were added over a solution of the corresponding
alkene 1a-h (1 mmol, 0.66 M) in acetonitrile (1.5 mL). The
suspension was shaken at 250 rpm for 24 h at 30 ºC, and then the
reaction was quenched by addition of water (1.5 mL). The
mixture was extracted with EtOAc (3 x 1.5 mL), and an aliquot
of the reaction was injected in the GC for the measurement of the
conversion values.
a Conversion values into epoxides 2k or 2l determined by GC.
3. Conclusions
In summary, a chemoenzymatic approach for the epoxidation
of alkenes has been studied using the lipase from Rhizomucor
miehei. Until now, this enzyme has been scarcely reported in the
literature for this type of indirect epoxidation reactions, probably
because of the versatility of Candida antarctica lipase B.
Reaction parameters have been optimized finding adequate
conditions for the recovery of a series of epoxides in moderate to
excellent yields. Thus, acetonitrile as solvent, lauric acid as
peracid precursor and the urea-hydrogen peroxide as oxidant
were found to be compatible with the use of RML. Significantly,
the selective epoxidation of limonene and (S)-carvone were
investigated using both RML and CAL-B, finding a complete
preference for the oxidation of the exocyclic alkene bond in the
case of the (S)-carvone and for the internal alkene bond in the
case of the limonene demonstrating the potential of enzymes,
4.4. General procedure for the epoxidation of 1,4-
dihydronaphtalene (1d) at 10 mmol scale.
Lauric acid (20 mmol, 4.01 g), RML (500 mg) and finally UHP
(11.1 mmol, 1.04 g) were added over a solution of 1,4-
dihydronaphtalene (1d, 10 mmol, 0.66 M) in acetonitrile (15
mL). The suspension was stirred for 24 h at 30 ºC. After this time
the reaction was quenched by addition of water (15 mL), and the
mixture extracted with EtOAc (3 x 15 mL).
Acknowledgments
We thank Novozymes for the generous gift of Rhizomucor
miehei lipase (RML) and Candida antarctica lipase type B
(CAL-B). Financial support from the Spanish Ministerio de
particularly hydrolases
transformations.
for
selective
chemoenzymatic
Ciencia
e
Innovación
(MICINN-12-CTQ2011-24237),
Principado de Asturias (SV-PA-13-ECOEMP-42) and the
University of Oviedo (UNOV-13-EMERG-01) are also gratefully
acknowledged.
4. Experimental section
4.1. General considerations.
References
Rhizomucor miehei lipase (150 U/g) and Candida antarctica
lipase type B (7600 PLU/g) were a gift from Novozymes.
Chemical reagents were purchased from different commercial
sources and used without further purification. Solvents were
distilled over an adequate desiccant under nitrogen. Flash
chromatographies were performed using silica gel 60 (230-240
mesh). 1H and 13C NMR experiments were obtained using a
Brüker AV-300 spectrometer (1H, 300.13 MHz and 13C, 75.5
MHz).
1. (a) Matsumoto, K.; Katsuki, T.; Arends, I. W. C. E in Science of
Synthesis , Stereoselective Synthesis, ed. de Vries, J. G.; Molander,
G. A. and Evans, P. A. Thieme, 2011, 1, 69-121; (b) Urlacher, V.
B.; Girhard, M. Trends. Biotechnol. 2012, 30, 26-36; (c) Hauser,
S. A.; Gokoja, M.; Kühn, F. E. Catal. Sci. Technol. 2013, 3, 552-
561;
2. See for instance: (a) Sharpless, K. B. Angew. Chem. Int. Ed. 2002,
41, 2024-2032; (b) Arends, I. W. C. E. Angew. Chem. Int. Ed.
2006, 45, 6250-6252; (c) Díez, D.; Nunez, M. G.; Antón, A. B.;
Moro, R. F.; Garrido, N. M.; Marcos, I. S., Basabe, P.; Urones, J.
G., Curr. Org. Synth. 2008, 5, 186-216; (d) Shi, Y. in Modern
Oxidation. Methods, ed. J.-E. Bäckvall, Wiley-VCH, Weinheim,
2010, pp 85-115; (e) Roiban, G.-D.; Agudo, R.; Reetz, M. T.,
4.2. Analytical conditions for determination of conversion values.

5
Tetrahedron 2013, 69, 5306-5311; (f) Li, A.; Liu, J.; Pham, S. Q.;
Li, Z. Chem. Commun. 2013, 49, 11572-11574.
ACCEPTED MANUSCRIPT
3. (a) Modern Biooxidation. Enzymes, Reactions and Applications,
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