Aerobic oxidation of isosorbide and isomannide employing TEMPO/laccase

Article abstract of DOI:10.1039/c3gc41855c

The oxidation of the renewable diols isosorbide and isomannide was successfully achieved using a TEMPO/laccase system. Furthermore, various TEMPO-derivatives were tested leading to conversions of up to >99% for the oxidation of isosorbide, isomannide, indanol and a halohydrin to the corresponding ketone. the Partner Organisations 2014.

Full text of DOI:10.1039/c3gc41855c

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Aerobic oxidation of isosorbide and isomannide
employing TEMPO/laccase†
Johannes Gross,a Katharina Tauber,a Michael Fuchs,a Nina G. Schmidt,a
Aashrita Rajagopalan,a Kurt Faber,a Walter M. F. Fabian,a Jan Pfeffer,b Thomas Haasb
and Wolfgang Kroutil*a
Cite this: Green Chem., 2014, 16,
2117
Received 5th September 2013,
Accepted 22nd January 2014
DOI: 10.1039/c3gc41855c
www.rsc.org/greenchem
The oxidation of the renewable diols isosorbide and isomannide interest as starting materials for pharmaceutical applications
was successfully achieved using a TEMPO/laccase system. Further- as well as for organic solvents or fuels and for biopolymers.3,4
more, various TEMPO-derivatives were tested leading to conver- For instance, recently, 1 was derivatised to the corresponding
sions of up to >99% for the oxidation of isosorbide, isomannide, diamine as a building block for polymers,5 or oxidised by
indanol and a halohydrin to the corresponding ketone.
chemical means.6 Amination of 1 by a ADH/transaminase bio-
cascade led to only a low amount of mono-amination.7
Especially the transformation of isosorbide to valuable deriva-
tives by functionalization or substitution of both hydroxyl
groups is difficult due to steric hindrance and different reactiv-
ities of the two hydroxyl groups.
The diols isosorbide 1 and isomannide 2 (Scheme 1) are both
gained from renewable resources like cellulose1 and starch.2
They represent platform chemicals for the potential replace-
ment of oil-based products.3 Consequently, they are in focus of
Results and discussion
Alcohol oxidation employing alcohol dehydrogenases
(ADHs)7,8 represents one possible common biocatalytic
method to prepare ketones or aldehydes from the corres-
ponding alcohols.9 Consequently, various ADHs were investi-
gated to oxidise isosorbide 1 as well as isomannide 2 to the
corresponding diketone 5. However, the levels of conversion
were unsatisfactory.
A maximum of 4% conversion was
reached at 50 mM substrate concentration over 24 hours. Cal-
culating the required Gibbs free energy for the oxidation
revealed that the energy needed for a mono-oxidation to the
hydroxy ketone followed by hydration to the favoured hydrate
(3a, 4a) is rather high (Scheme 1), even higher than for the dis-
favoured biocatalytic oxidation of halohydrins by ADHs
(4–6 kcal mol−1).10 For instance, for the oxidative hydrogen
transfer of isosorbide 1 to the exo-hydroxy ketone 3 6.7 kcal
mol−1 are required while 8.9 kcal mol−1 are needed to get the
endo-hydroxy ketone 4. Considering the hydrated forms (3a,
4a), the energy decreases by approximately 1 kcal mol−1, still
representing a significant burden for the hydrogen transfer to
yield high amounts of ketone. The higher energy needed to get
the endo-hydroxy ketone 4 can be explained by the higher
steric hindrance of the hydride to be removed at the exo-
hydroxy of 1. Also for the second oxidation (transformation of
3 and 4, respectively, to diketone 5 and its hydrate 5a) a signifi-
cant energy barrier has to be overcome. To address the
Scheme 1 Relative Gibbs free energies (kcal mol−1) for the oxidation
and hydration of isosorbide 1 and isomannide 2. Energies for oxidations
were calculated for the reaction of the alcohol with acetone yielding the
ketone plus 2-propanol. The calculations were performed using the
MP2/cc-pVTZ//MP2/cc-pVDZ procedure in aqueous solution (IEFPCM
solvation model).
aDepartment of Chemistry, Organic and Bioorganic Chemistry, University of Graz,
Heinrichstrasse 28, A-8010 Graz, Austria. E-mail: Wolfgang.Kroutil@uni-graz.at;
Fax: +43 316 380 9840; Tel: +43 316 380 5350
bEvonik Industries AG, CREAVIS Technologies & Innovation, Paul-Baumann-Strasse
1, 45772 Marl, Germany
†Electronic supplementary information (ESI) available: Analytics, sources of
chemical and enzymes, computational details. See DOI: 10.1039/c3gc41855c
This journal is © The Royal Society of Chemistry 2014
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problem of steric hindrance for the oxidation of the exo-
hydroxy moiety as well as to provide sufficient energy, an oxi-
dation method was needed involving a small mediator as an
oxidizing agent and a high energy terminal oxidant like mole-
cular oxygen. In case of success, the same approach could be
applied for the oxidation of compounds 6–8, which are also
difficult susbtrates for ADHs.
Consequently, the combination of laccase and TEMPO M1
as a mediator was tested employing O2 as an oxidant
(Scheme 2).11 Laccases12 can be produced in large quantities,13
and the oxidation system can be employed at ambient temp-
erature (20–30 °C) and in aqueous medium. Although TEMPO
has been employed for the oxidation of various sec-alcohols
using various chemical reoxidation reagents,14 sec-alcohols
have only been sparsely oxidised with the TEMPO/laccase
system.15 TEMPO enables an aerobic green oxidation.16
In a first attempt, commercial laccases from Trametes versi-
color (TvLac) and Agaricus bisporus (AbLac) were applied using
sodium acetate buffer at varied pHs or in deionised water
(Table 1). Best results were achieved with TvLac while AbLac
led to very low conversions. Based on the published sequences
of laccases from these fungi, they are similar in size (TvLac:
519aa, AbLac: 520aa), but have only 47.5% sequence identity.17
A redox potential is only published for TvLac (E0′ = 785 mV vs.
normal hydrogen electrode).18 TEMPO/TvLac oxidised the two
equal hydroxy groups of isomannide 2 as well as the two ener-
getically different hydroxy groups of isosorbide 1 to the di-
ketone 5 at pH 5.5 and 6.5. For 1 both possible intermediates –
the endo- and the exo-hydroxyyketone – were observed at pH
4.5, indicating that in the first oxidation step no absolute dis-
crimination between the two hydroxy moieties occurred; for 2
obviously only the endo-hydroxyketone can be formed due to
the stereochemistry of the substrate. Blank experiments
showed that in the absence of an enzyme or a mediator no
product formation was observed. Interestingly, just using water
without buffer salts (last entry, pH 6.5) led to perfect con-
version, offering another opportunity to minimize waste.
Table 1 Oxidation of isosorbide 1 and isomannide 2 applying TEMPO/
laccasea
endo-OH- exo-OH-
ketone 4
ketone 3 Diketone
Substr. Buffer
pH Laccaseb [%]
[%]
5 [%]
1
1
1
1
1
1
1
1
2
2
2
2
NaOAc
NaOAc
NaOAc
5.5 None
4.5 AbLac
5.5 AbLac
<0.1
<0.1
1
2
8
<0.1
<0.1
<0.1
4
23
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
13
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
79
>99
>99
<0.1
<0.1
77
H2O dist. 6.5 AbLac
NaOAc
NaOAc
NaOAc
NaOAc
NaOAc
NaOAc
NaOAc
4.5 TvLac
5.5 TvLac
6.5 TvLac
5.5 TvLacc
4.5 AbLac
4.5 TvLac
5.5 TvLac
>99
>99
H2O dist. 6.5 TvLac
a Relative amounts as peak areas measured by GC-FID. Substrate
(50 mM), laccase (10 U), TEMPO (30 mol%), 24 h at 30 °C, 120 rpm.
b TvLac = Laccase from Trametes versicolor; AbLac = Agaricus bisporus.
c In the absence of TEMPO.
Isomannide 2 was in general transformed faster than 1.
Although these first tests gave promising results, optimisation
studies were required to decrease the concentration of the
mediator and the enzyme.
In the next experiments the TEMPO concentration was
lowered to 5 mol%. In two buffer systems (acetate and phos-
phate) the highest conversion was achieved at low buffer con-
centration (Fig. 1). In general, best results were obtained at pH
6 and low buffer concentrations (5–10 mM).
Interestingly, for bis-tris buffer and sodium citrate buffer
conversion did not exceed 3%, although oxidation of glycols
and various alcohols is reported using sodium citrate buffer at
a pH of 4.5 and high buffer concentrations.15c Obviously these
conditions are not favourable for isosorbide or isomannide as
a substrate. Further decrease of the buffer concentration to
0.5–1.5 mM improved the conversion and led to full diketone
formation as was observed in the deionised water-system.
To minimize the concentration of the mediator, the TEMPO
concentration was decreased successively. Reducing the
TEMPO concentration from 30 mol% to
4 mol% (mol
mediator/mol substrate) did not lead to a significant decrease
of the diketone formation employing isosorbide 1 as the sub-
strate over 24 hours under the conditions employed. However,
as soon as the mediator concentration was below 4 mol%, con-
version decreased rapidly (Fig. 2).
To identify the most suitable mediator,15a,19 a selection of
different mediators M2–M15 were tested (Fig. 3).
However, for isosorbide 1 as a substrate, TEMPO M1 was
superior to all other TEMPO-derivatives M2–M15.
Investigating the substrate scope of the oxidation method,
also 1-indanol 6 was tested. Using 6 as a substrate, TEMPO M1
led to 45% conversion at 50 mM substrate concentration
within 4 hours. For this substrate, a number of mediators
proved to be superior compared to M1 (Fig. 4): for instance,
M3 led to 99% conversion, M4 gave 65% conversion, M8 76%,
Scheme 2 Oxidation of isosorbide 1 employing TEMPO/laccase and
further substrates investigated.
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Fig. 3 Selection of mediators: TEMPO and derivatives.
Fig. 1 Conversions at varied pH in acetate buffer (A) and phosphate
buffer (B) (5 mM, 10 mM and 50 mM) using 1 as a substrate. Conversions
were measured by GC-FID. Reaction conditions: TEMPO (5 mol%), iso-
sorbide (50 mM), TvLac (0.5 mg ml−1); 24 h at 30 °C at 120 rpm. Lines
between the measured points are only drawn as a guide for the eye to
ease reading and not to interpolate values.
Fig. 4 Oxidation of 1-indanol 6 (50 mM) employing different mediators
(5 mol%) in phosphate buffer (pH 6, 5 mM) employing TvLac (0.5 mg
ml−1); 4 h at 30 °C, 120 rpm. Conversions were measured by GC-FID.
and mediator M15 75%. It is noticeable that all these
mediators possess a carboxylic acid moiety. Mediator M2
(41%) and mediator M14 (42%) led to results comparable to
TEMPO M1.
Increasing the mediator concentration of TEMPO M1 to
10 mol%, 1-indanol 6 was quantitatively oxidised within
24 hours (Fig. 5). In contrast, the halohydrin 2-chloro-1-phenyl
ethanol 7 was oxidised only efficiently in the presence of elev-
ated TEMPO concentration (45 mol%) giving 87% conversion
after 24 hours.
While indanol (50 mM) could be oxidised using M3 as a
mediator at only 2.5 mol% with full conversion to the corres-
ponding ketone within 24 hours, substrate 7 led with M3 only
to 4% conversion under the same conditions. On the other
hand, 1,2,3,4-tetrahydronaphthalen-1-ol 8 was oxidised with
88% with 2.5 mol% M3. The results leading to highest conver-
sions are summarized in Table 2.
Fig. 2 Variation of TEMPO concentration in phosphate buffer (0.5, 2.5
and
5 mM, pH 6) using isosorbide 1 as a substrate. Conversions
measured by GC-FID. Reaction conditions: substrate (50 mM); TvLac
(0.5 mg ml−1), 24 h at 30 °C, 120 rpm. Lines between the measured
points are only drawn as a guide for the eye to ease reading and not to
interpolate values.
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work-up the samples were lyophilised, extracted with acetone
(1 ml) and centrifuged (13 000 rpm, 3 min at room tempera-
ture). The organic layer was dried (Na2SO4) and analysed on
GC-FID. Products were identified by comparison with auth-
entic reference material.
Reaction conditions for alcohols 6–8
TEMPO/acetone-solution (15 µl, mediator concentration in the
reaction: 2.5 mM) was transferred into Sarstedt tubes (15 ml)
and acetone was evaporated. Then phosphate buffer (980 µl,
2.5 mM, pH 6) as well as the substrate (50 mM) was added and
the oxidation was started by addition of the enzyme solution
(20 µl, end concentration 0.5 mg ml−1). Reactions took place at
30 °C and 120 rpm on a rotatory shaker. For work-up the
samples were extracted with ethyl acetate (2 × 500 µl) and cen-
trifuged (13 000 rpm, 3 min at room temperature). The organic
layer was dried (Na2SO4) and analysed on GC-MS. Products
were identified by comparison with authentic reference
material.
Fig. 5 Variation of the concentration of M1 in phosphate buffer (pH 6,
5 mM) for the oxidation of 1-indanol 6 and chloroacetophenone 7
(50 mM). Conversions were measured by GC-FID. Conditions: TvLac
(0.5 mg ml−1), 24 h at 30 °C, 120 rpm.
Table 2 Optimised conditions for oxidationa
Substr.
Mediator
Mediator conc. [mol%]
Conv. [%]
Acknowledgements
1
2
6
7
8
TEMPO M1
TEMPO M1
M3
M3
M3
5
5
2.5
45
2.5
>99b
>99b
>99
87
The research leading to these results has received funding
from the German Federal Ministry of Education and Research
(BMBF) within the project “Bioxamine” (grant no. 0316044B)
and Evonik Industries AG. Some TEMPO derivatives were pro-
vided by BASF Schweiz AG.
88
a Conditions: 50 mM substrate, laccase (10 U), phosphate buffer
(5 mM, pH 6) 24 h, 30 °C, 120 rpm. b >99% of diketone 5 were
obtained.
Notes and references
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Full conversions were achieved for the di-oxidation of the
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system. Best results were obtained for instance in phosphate
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