38
A. Drożdż et al. / Catalysis Communications 81 (2016) 37–40
Sigma Aldrich. Novozyme-35 was donated by Novozymes. Enzym
MsAcT was produced and the activity determined as described earlier
[14].
2.2. General method for Baeyer-Villiger oxidation
The ketone (0.25 mmol) and 0.5 ml of ethyl acetate (5.09 mmol)
were introduced into a 25 ml round-bottom flask and the contents of
the flask was shaken. Next, 4 mg of MsAcT was introduced, and 60%
aq. H2O2 (0.50 mmol) was added dropwise. The flask was sealed with
a septum and mixed in a thermostated shaker ( 0.5 °C) with orbital
stirring at 250 rpm at 35 °C for 2 h to 5 days, depending on the reaction
rate. Periodically, 10 μl of the sample diluted with 0.7 ml of dichloro-
methane was collected during the reaction to monitor the progress of
the reaction utilising GC (Perkin Elmer Clarus 500 chromatograph
with SUPELCOWAX™ 10 column (30 m × 0.2 mm × 0.2 μm) with n-
decane as an external standard.
The structure of the products were confirmed using GC–MS analysis
(Agilent Gas Chromatograph 7890C equipped with a HP-5 MS column
(30 m × 0.25 mm × 0.25 μm; MS Agilent 5975C, EI ionization 70 eV,
and the results were compared to NIST/EPA/NIH Mass Spectral Library.
Fig. 1. Effect of MsAcT content on the BV oxidation of 2-methylcyclohexanone
(0.25 mmol) with 30% aq. H2O2 (0.50 mmol) in ethyl acetate (5.09 mmol, 0.5 ml) at 25 °C.
3. Results and discussion
The effect of the buffer was also checked for two forms of CALB li-
pase; a native (liquid) and its immobilized form - Novozyme 435. As
can be seen from Fig. 3 the presence of water appeared to exert a strong
negative effect on their activity, regardless the biocatalyst form. This
could be ascribed to two factors: (i) specific structure of the lipase, espe-
cially the presence of hydrophobic polypeptide chain (lid or flat), isolat-
ing its active centre from the reaction medium [17], (ii) hydrolysis of
ethyl acetate which lowered the pH and this brought the reaction to a
halt.
Further studies aimed to determine the effect of oxidising agent (30
and 60% aq. H2O2, urea hydrogen peroxide UHP). They showed that the
most reactive is 60% aq. H2O2 (Fig. 4). A twofold molar ratio of 60% aq.
H2O2 to the ketone was large enough to ensure optimum kinetics, i.e.
very similar in value to that obtained using a fourfold excess of 30%
aq. H2O2. It is noteworthy, that the idea of using 60% aq. H2O2 was also
aimed to probe the MsAcT performance under harsh reaction condi-
tions, since the exposure of CALB to high concentration of aq. hydrogen
peroxide resulted in its complete deactivation [18]. Anhydrous UHP ap-
peared to be poorly reactive under these reaction conditions. The efforts
for the isolation of enzyme after the reaction were unsuccessful. The use
of enzyme immobilization methods may produce improvements in the
enzyme performance, as was already described in the literature. [19,20].
As can be seen from Scheme 1 the applied chemo-enzymatic method
of lactone synthesis involves MsAcT as the biocatalyst of peracid forma-
tion, hydrogen peroxide as oxidant and ethyl acetate as both the peracid
precursor and solvent. As a model ketone 2-methylcyclohexanone was
used.
The experiments were performed under the reaction conditions rec-
ommended for this reaction (25 °C, molar ratio of ketone to 30% aq.
H2O2 1: 2) [6]. At first, they aimed at discriminating the regions of spe-
cific kinetic control of the chemo-enzymatic Baeyer-Villiger reaction.
They were made by varying the amount of MsAcT in the range of
2–7 mg, while keeping the amount of a model ketone constant
(0.25 mmol; 2-methylcyclohexanone). For a fixed value of molar ratio
of the ketone to 30% aq. H2O2 (1:2) and an excess of ethyl acetate the
rate of lactone formation at 25 °C appeared to depend on the biocatalyst
content, provided it was ≤4 mg (Fig. 1). Those findings delineated the
region of effective control of ketone oxidation by the created peracid
(4–7 mg of MsAcT). Since differences in the reaction courses carried
out using 4 and 7 mg of the enzyme per 0.25 mmol of ketone appeared
to be small, therefore all further studies were performed using 4 mg of
the enzyme.
At this stage we also checked the influence of ethyl acetate and the
possibility to replace its excess with a buffer. These studies showed
that the 5.09 mmol of ethyl acetate (0.5 ml) per 0.25 mmol of ketone
(approximately, only 5% of ethyl acetate is consumed for the reaction)
is the most effective and that the addition of the buffer has insignificant
influence on the reaction rate (Fig. 2). The latter phenomenon could be
explained by an extensive hydrophobicity of MsAcT active center [11,
13–16]. In all cases, with the addition of buffer or not, the reaction sys-
tem was always biphasic according to the use of water solution of H2O2
and the creation of water molecule as a by-product in the reaction.
Fig. 2. The influence of the solvent on the BV oxidation of 2-methylcyclohexanone
Scheme 1. Model chemo-enzymatic Baeyer-Villiger oxidation studied in this work.
(0.25 mmol) with 30% aq. H2O2 (0.50 mmol) and MsAcT (0.004 g) at 25 °C.
Drozdz
