Tailored sol-gel immobilized lipase preparates for the enzymatic kinetic resolution of heteroaromatic alcohols in batch and continuous flow systems

Article abstract of DOI:10.1039/c7ra10157k

Tailored immobilized lipases from Candida antarctica B and Pseudomonas fluorescens, with improved thermal and operational stability, were prepared through fine tuning of the structure of the sol-gel matrix, using various binary or ternary precursor mixtures for the EKR of various chiral heteroaromatic secondary alcohols with benzofuran, benzo[b]thiophen, phenothiazine and 2-phenylthiazol moieties. The operational stability in batch process was studied for five selected systems by performing reuse experiments, using the conversion, enantiomeric excesses and enantiomeric ratio as parameters, demonstrating the dependence of the sol-gel lipase preparate performance on the structure of both biocatalyst and substrate. The resolution of the benzofuranic substrates with the best performing biocatalysts was studied in continuous-flow mode, using the productivity as a criterion. The specific reaction rates under continuous-flow operation (r_flow) were higher than those obtained in batch mode (r_batch) in both cases, sustaining its usefulness for further process development.

Full text of DOI:10.1039/c7ra10157k

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Tailored sol–gel immobilized lipase preparates for
the enzymatic kinetic resolution of heteroaromatic
alcohols in batch and continuous flow systems†
Cite this: RSC Adv., 2017, 7, 52977
a
a
b
Madalina Elena Moisa,
˘ ˘
Cristina Georgiana Spelmezan,
Cristina Paul,
˘
a
a
a
Judith Hajnal Bartha-Vari,
Laszlo Csaba Bencze,
Florin Dan Irimie,
´
´
´
a
b
a
*
Csaba Paizs,
Francisc Peter
and Monica Ioana Tosa
¸
´
´
Tailored immobilized lipases from Candida antarctica B and Pseudomonas fluorescens, with improved
thermal and operational stability, were prepared through fine tuning of the structure of the sol–gel
matrix, using various binary or ternary precursor mixtures for the EKR of various chiral heteroaromatic
secondary alcohols with benzofuran, benzo[b]thiophen, phenothiazine and 2-phenylthiazol moieties.
The operational stability in batch process was studied for five selected systems by performing reuse
experiments, using the conversion, enantiomeric excesses and enantiomeric ratio as parameters,
demonstrating the dependence of the sol–gel lipase preparate performance on the structure of both
biocatalyst and substrate. The resolution of the benzofuranic substrates with the best performing
biocatalysts was studied in continuous-flow mode, using the productivity as a criterion. The specific
reaction rates under continuous-flow operation (rflow) were higher than those obtained in batch mode
(rbatch) in both cases, sustaining its usefulness for further process development.
Received 12th September 2017
Accepted 9th November 2017
DOI: 10.1039/c7ra10157k
rsc.li/rsc-advances
immobilization, the protein is multipoint attached to a carrier
by covalent bonds, directly or through a spacer. One of the
Introduction
benets of this method is that enzyme leaching is not possible,
but the activity could be signicantly modied, decreasing in
most of the cases.
With the aims of stabilizing enzymes and improving enzymatic
activity and selectivity, the development of effective immobili-
zation methods has become crucial for the synthetic applica-
tions of lipases. Moreover, the processes performed with
immobilized catalysts offer as advantages continuous produc-
tion, the absence of the biocatalyst in the product stream and
the possibility to reuse the biocatalyst. In this way, the synthetic
and production processes could be more selective and are for
certain more procient economically and environmentally.
Accordingly, immobilization methods such as binding to
a carrier, cross-linking, and encapsulation in an organic or
inorganic polymeric matrix have been intensively studied.1–8
While the adsorption on solid supports (such as Celite, a dia-
tomaceous earth) is a simple, inexpensive and easy to perform
method, suitable for applications in organic solvents, the
enzyme leaching from the support is generally the most
important disadvantage of this procedure.9 In the covalent
For enzymes which are easily deactivated (for instance by
covalent immobilization), encapsulation could be the most
useful method. In the case of sol–gel encapsulation inert glasses
with improved catalytic properties (porosity, thermal and
mechanical stability, and tunable hydrophobicity) are prepared.
The synthesis can be performed by acid- or base-catalyzed
hydrolysis of the organic silane precursors, followed by
condensation of the resulted intermediates in the presence of
an enzyme.10–12 When some protective additives (crown ether,
isopropyl alcohol, etc) were used in the sol–gel immobilization
process, for certain substrates a supplementary increase of the
lipase activity in esterication and enzymatic kinetic resolution
(EKR) processes was already reported.13–15
Lipases are most frequently used for the synthesis of chiral
intermediates by kinetic resolution in an immobilized form.16,17
Using continuous-ow reactors, the performances of
enzyme-catalyzed reactions are generally signicantly
improved, including the reduction of reaction time, as
compared to the classical batch method. The packed-bed
reactor (a column containing the immobilized biocatalyst
which is ushed with the reaction mixture using a pump,
allowing ow and residence time control) is usually the rst and
simplest choice.18–21
aBiocatalysis and Biotransformation Research Center, Faculty of Chemistry and
´
Chemical Engineering, Babe¸s-Bolyai University of Cluj-Napoca, Arany Janos 11,
RO-400028, Cluj-Napoca, Romania. E-mail: mtosa@chem.ubbcluj.ro
bBiocatalysis Group, Faculty of Industrial Chemistry and Environmental Engineering,
University Politehnica Timi¸soara, C. Telbisz 6, RO-300001, Timi¸soara, Romania
† Electronic supplementary information (ESI) available: Protocols for lipases
immobilization, chemical synthesis of racemic compounds, chromatographic
chiral separation conditions, lipase and solvents screening results, recyclability
studies results. See DOI: 10.1039/c7ra10157k
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In the present research, several immobilized lipase pre-
The silane precursors, methyl- (MeTMOS), vinyl- (VTMOS),
parates (lipases from Candida antarctica B and from Pseudo- octyl- (OcTMOS), and phenyl-trimethoxysilane (PhTMOS) were
monas uorescens immobilized by sol–gel entrapment, purchased from Merck (Germany), excepting tetra-methoxysilane
adsorption on Celite or their combination) were tested in (TMOS), a product of Fluka. The other materials and additives
transesterication reactions of various heteroaromatic used for immobilization, tris-(hydroxymethyl)-aminoethane, iso-
secondary alcohols, useful chiral building blocks for the phar- propyl alcohol, Celite 545, acetone and hydrochloric acid were
maceutical industry.22–25 Previous studies15,26–28 already stated products of Merck, while sodium uoride and the ionic liquid
the versatility of the sol–gel immobilization method and the 1-octyl-3-methyl-imidazolium tetrauoroborate [Omim]BF4 were
possibility to ne tune the composition of the binary or ternary purchased from Fluka.
precursors mixture to achieve high activity and selectivity, but
Vinyl acetate and the solvents used for enzymatic reactions
these studies were carried out on model secondary alcohols were of the highest grade from Sigma-Aldrich or Merck and
˚
(1-phenylethanol or 2-alkanols). The present study demon- stored over molecular sieves (4 A). Solvents employed in
strates for the rst time the efficiency of the tailored binary and chemical synthesis were used as purchased (methanol > 99.8%)
ternary sol–gel immobilized lipase preparates for the EKR of or dried over molecular sieve (dichloromethane) or sodium
various heteroaromatic alcohols. The recycling capacity of the (diethyl ether). Other reagents were purchased from Sigma-
best performing biocatalysts was also investigated and two Aldrich or Merck.
continuous-ow procedures were developed by testing the
A Heidolph Vibramax 110 shaker equipped with incubator
most important reaction parameters: temperature, substrate module (Heidolph, Germany) was used for all batch experiments.
concentration, and ow rate.
Continuous-ow experiments were performed in a stainless
steel column (50 ꢂ 2.1 mm) using the thermostat and the pump
modules of an Agilent 1200 Series HPLC instrument.
Experimental
Materials and methods
Chemical synthesis
The synthesis of the investigated racemic substrates and prod-
ucts was rst performed using known methods29–33 from the
corresponding prochiral heteroaryl-methyl-ketones (1a–c,f) by
chemical reduction with sodium borohydride or from the car-
baldehydes (1d,e,g–i) by Grignard reaction. Racemic ethanols
rac-2a–i were next transformed with acetyl chloride (1.2 equiv.)
and a catalytic amount of 1% DMAP/pyridine in dry CH2Cl2 into
the corresponding racemic acetates rac-3a–i (Scheme 1a). The
obtained NMR data for rac-2,3a–i are in agreement with those
already reported.29–33
Thin layer chromatography (TLC) was performed on Merck
Kieselgel 60F254 sheets. Spots were visualized by treatment
with 5% ethanolic phosphomolybdic acid solution followed by
heating. Preparative chromatographic separations were per-
formed using column chromatography on Merck Kieselgel 60
(63–200 mm).
1H-NMR and 13C-NMR were recorded in CDCl3 at 25 ꢀC using
a Bruker spectrometer operating at 300 or 600 MHz and 75 or
151 MHz, respectively.
Chiral HPLC (Agilent 1200 Series instrument) and GC (Agi-
lent 77890A instrument) were employed for the quantitative
analysis of the enzymatic kinetic resolutions, by periodically
taking samples (50 mL) and analyzing them by chiral chroma-
tography: GC (compounds rac-2,3a,b) or HPLC (compounds rac-
2,3c–i). The type of chiral columns used for the enantiomeric
separations, separation conditions and retention times for each
individual enantiomer are presented in Table 1S.† The absolute
conguration of products was assigned according to previously
reported data, based on the retention time of a reference
enantiomerically enriched compound.29–33
The enantiomeric excesses of the substrate eeS and product
eeP were determined from peak areas of GC or HPLC chro-
matograms. The enantiomeric ratio (E) was calculated with the
known equation E ¼ ln[(1 ꢁ c)(1 ꢁ eeS)]/ln[(1 ꢁ c) (1 + eeS)],
using the calculated conversion c ¼ eeS/(eeS + eeP).34
Lipase B from Candida antarctica was purchased as solid
lyophilized powder from C-Lecta (Leipzig, Germany, product
code 20606-1). Lipase AK (beige-brown powder, from Pseudo-
monas uorescens) and lipase A from Candida antarctica were
bought from Sigma-Aldrich (Germany, product code 534730
and 65986 respectively). Novozyme 435 (lipase from Candida
antarctica B, immobilized on acrylic resin) was also purchased
from Sigma Aldrich.
Immobilization of lipases
The immobilization protocols for the sol–gel entrapment of
lipases B from Candida antarctica and Pseudomonas uorescens
(Amano-AK) were accomplished as previously described
(Table 1), with NaF as catalyst for simultaneous hydrolysis and
polycondensation (Method 1)27 or using HCl for the formation
of a prepolymer (Method 2).35 The combined method of sol–gel
entrapment and adsorption was also carried out as described
elsewhere.26–28 Method 3 was simple adsorption on Celite 545
(see the ESI† for detailed description of all immobilization
Scheme 1 (a) Chemical synthesis of racemic substrates; (b) EKR
mediated by lipases.
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Table 1 Immobilized lipases description
Preparate codea
Immobilization method
Silane precursorsb (molar ratio)
CaL-B 1 BS
CaL-B 1 BSC
CaL-B 1 TS
CaL-B 1 TSC
CaL-B 1 TS*
CaL-B 2 TS
CaL-B 2 TSC
CaL-B Celite
AK 1 BS
Method 1
Method 1 + adsorption on Celite 545
Method 1
Method 1 + adsorption on Celite 545
Method 1
Method 2
Method 2 + adsorption on Celite 545
Method 3 (adsorption on Celite 545)
Method 1
VTMOS : TMOS (4 : 1)
VTMOS : TMOS (4 : 1)
PhTMOS : VTMOS : TMOS (1.6 : 0.4 : 1)
PhTMOS : VTMOS : TMOS (1.6 : 0.4 : 1)
PhTMOS : MeTMOS : TMOS (1.6 : 0.4 : 1)
PhTMOS : MeTMOS : TMOS (1.6 : 0.4 : 1)
PhTMOS : MeTMOS : TMOS (1.6 : 0.4 : 1)
—
OcTMOS : TMOS (1 : 1)
AK 1 BSC
AK 1 TS
AK 1 TSC
AK 2 TSC
Method 1 + adsorption on Celite 545
Method 1
Method 1 + adsorption on Celite 545
Method 2 + adsorption on Celite 545
OcTMOS : TMOS (1 : 1)
PhTMOS : VTMOS : TMOS (1.6 : 0.4 : 1)
PhTMOS : VTMOS : TMOS (1.6 : 0.4 : 1)
PhTMOS : MeTMOS : TMOS (1.6 : 0.4 : 1)
a
b
Lipases source: CaL-B-lipase B from Candida antarctica, AK-lipase from Pseudomonas uorescens. VTMOS (vinyltrimethoxysilane), PhTMOS
(phenyltrimethoxysilane), MeTMOS (methyltrimethoxy-silane), OcTMOS (octyltrimethoxysilane) and TMOS (tetramethoxysilane).
methods). The protein loading values, calculated for 1 g bio- temperatures (25–65 ꢀC) at ow rates of 0.1–0.5 mL minꢁ1. The
catalyst, were 17.4 mg for sol–gel entrapment and adsorption on residence time (RT) was calculated using the volume of n-
Celite and 10.7 mg for the entrapment combined with adsorp- hexane in the lipase-lled reactor (measuring the weights of the
tion, respectively. They were determined as described previ- dry and wet lipase-lled column) and the ow rate. Samples (50
ously.27 The immobilization method and the silane precursors mL) were taken periodically, diluted with n-hexane (500 mL) and
used for every preparate selected for the present study are analyzed by HPLC. The steady-state condition was reached in
described in Table 1.
30–90 min, depending on the used ow. Between experiments,
the lipase-lled column was washed with n-hexane at 0.5
mL minꢁ1 for 20 min and stored overnight at 4 ꢀC.
Enzymatic O-transesterications
Lipase-catalyzed O-acylation of rac-2c,d in batch mode. To
a solution of rac-2c,d (10 mmol) in n-hexane (1 mL), vinyl acetate
(0.75 equiv. and 3 equiv., respectively) and the immobilized
lipase (5 mg) were added and shaken at 700 rpm at 65 ꢀC until
the maximum conversion was reached (48% aer 14 h for rac-2c
and 50% aer 2.5 h for rac-2d). Samples were collected as
previously mentioned and subjected to HPLC analysis.
All experiments were performed in triplicate and the average
values were further used.
Analytical-scale lipase-mediated O-acylation of racemic
hetero-aryl-ethanols rac-2a–i. The immobilized lipase (5 mg for
the 17.4 mg protein per g biocatalyst preparates and 8 mg for
the 10.7 mg protein per g biocatalyst preparates), the tested
substrate (10 mmol), solvent (1 mL) and vinyl acetate (30 mmol,
2.77 mL) were combined in a reaction vial and shaken at 700 rpm
at 25 ꢀC until z50% conversion was reached. For the reactions
performed in absence of solvent, vinyl acetate was added in
excess (250 mL). Samples (50 mL) were taken periodically from
the reaction vials and diluted with 950 mL n-hexane/2-propanol
for HPLC analysis or 950 mL CH2Cl2 for GC analysis and then
ltered before analysis.
Reuse experiments. The rst cycle of enzymatic trans-
esterication was performed as described above. Aer each cycle
the enzyme was washed three times with 1 mL n-hexane, and
immediately reused in the next cycle. Samples for GC or HPLC
analysis were collected aer each cycle as previously mentioned.
Lipase-catalyzed O-acylation of rac-2c,d in continuous-ow
packed-bed microreactors. Continuous-ow experiments were
performed in a thermostated stainless steel column (50 ꢂ
2.1 mm) lled with immobilized lipase (107 mg of AK 1 BS and
51 mg of CaL-B 1 TS*) attached to an Agilent 1200 Series HPLC
pump. Before performing the reactions, the microreactor was
washed with n-hexane at 0.5 mL minꢁ1 for 20 min. The solution
Results and discussion
Chemical synthesis of racemic heteroaryl-ethanols rac-2a–i
and their corresponding acetates rac-3a–i
Racemic heteroaryl-ethanols rac-2a–c,f were obtained starting
from the corresponding prochiral heteroaryl-methyl-ketones
(1a–c,f) by chemical reduction with sodium borohydride,
while in the case of rac-2d,e,g–i the Grignard reaction of car-
baldehydes 1d,e,g–i as starting materials was used (Scheme 1a).
Racemic ethanols rac-2a–i were used next as substrates in
lipase-catalyzed transesterication reactions (Scheme 1b) and
also for the synthesis of the corresponding racemic acetates
rac-3a–i (see ESI,† Section 2), used for establishing the chro-
matographic separation methods (see ESI,† Section 3).
Enzymatic kinetic resolution of racemic ethanols rac-2a–i
through O-acylation with vinyl acetate and lipase preparates
in organic solvents
of the substrate rac-2c or rac-2d (10–100 mM) and vinyl acetate Efficient enzymatic procedures for the synthesis of enantio-
(0.75 equiv. or 3 equiv.) in n-hexane was pumped through the merically enriched (R)- and (S)-ethanols through enzymatic
lipase-lled
microreactor
thermostated
at
different kinetic resolution of the racemic ethanols or their esters
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(generally acetates) with commercially available lipases have investigated in several solvents with different polarities, such as
been already described.29–33
n-hexane, toluene, diisopropyl ether, methyl-tert-butyl ether,
An optimal immobilization method can improve the bio- dichloromethane and acetonitrile (see ESI, Tables 10S–18S†).
catalyst efficiency and the operational stability, allowing the The reactions were evaluated using the conversion c, enantio-
process operation in continuous ow reactors, which can meric excesses (of the untransformed alcohol eeS and of the
provide increased productivity. Lipase encapsulation in sol–gel formed ester eeP), and enantiomeric ratio E as parameters.
matrices leads to enzyme preparates with high mechanical and
As a preliminary general conclusion of the screening, all
thermal stability and also high porosity. Moreover the surface lipase preparates demonstrated high enantiomer selectivity
properties of the sol–gel matrix can be tailored for an optimal towards the racemic substrates in neat vinyl acetate, except
interaction of enzymes with particular substrates by choosing some L-AK preparates (AK 1 TSC and AK 2 TSC in the acylation
the appropriate silane precursors and synthesis method.
reaction of rac-2b, E ¼ 25–58 see Table 3S,† entries 14 and 15 in
The immobilizations were accomplished by adsorption on ESI†), while the conversion was inuenced by the structure of
Celite 545, sol–gel entrapment in mesoporous silica materials substrate, as well as by the immobilization method. A more
obtained from binary (BS) or ternary silane (TS) precursor detailed study of the inuence of different immobilization
mixtures, and sol–gel entrapment combined with adsorption on conditions was not the subject of this work.
Celite 545, as described in the experimental part (Table 1). Two
The optimal lipase preparates are summatively presented in
sol–gel entrapment methods were employed, one that uses NaF Table 2. Some particular information obtained for each
as catalyst for simultaneous hydrolysis and polycondensation substrate is discussed.
(Method 1) and another one that uses a sol prepolymer obtained
with HCl (Method 2).
The rst tested substrate, rac-1-(benzo[b]thiophen-2-yl)
ethan-1-ol rac-2a, was quantitatively resolved (c y 50%) with
The items used in the present work were selected from all tested CaL-B preparates and almost all L-AK preparates in
numerous preparates, according to their activities on model neat vinyl acetate (Table 2S in ESI†). In the given reaction
substrates (rac-2-hexanol and rac-1-phenylethanol, data not conditions, this substrate was easily acylated regardless to the
shown), with the aim to explore several immobilization tech- immobilization method, but it is noteworthy that the catalytic
niques based on entrapment and adsorption, as well as support efficiency of the immobilized preparates was higher (c ¼ 45–
matrices with different composition and morphology. In order 50%, E [ 200) compared to the reaction catalyzed by native
to be able to compare the catalytic activities of the tested lipase CaL-B, with only 16% conversion aer the same reaction time
preparates, the same enzyme content was used in all experi- (9.5 hours, as presented in Table 2S, entry 1 in ESI†). Since
ments. Vinyl acetate was employed as solvent and as irreversible almost all immobilized preparates studied in the acylation
acyl donor as the liberated vinyl alcohol undergoes spontaneous reaction of rac-2a gave very good results, for the solvent
tautomerization to acetaldehyde, shiing the reaction equilib- screening one candidate from each of the two forms of lipases
rium towards product formation.36
(CaL-B and AK) was selected (CaL-B 1 TSC and AK 1 TSC). The
In order to nd the possible applications of the tested lipase best results were obtained with CaL-B 1 TSC in n-hexane (c y
preparates in the chemoenzymatic synthesis of heteroaromatic 50% aer 1 h, see Table 10S, entry 1 in ESI†), therefore we tested
compounds, rst the selected substrates rac-2a–i (40 mM) were the other CaL-B preparates in the optimal solvent, n-hexane as
acylated in neat vinyl acetate in the presence of immobilized well. The results (Table 17S in ESI†) indicated that almost all
biocatalysts (see ESI, Tables 2S–9S†). Furthermore since the biocatalysts afforded maximum conversion of the substrate in
nature of the solvent could signicantly inuence the selectivity 1 h, except for CaL-B 1 BS (c ¼ 42%, Table 17S, entry 1 in ESI†)
and activity of the immobilized enzyme, the enzymatic acylation and CaL-B 2 TSC (c ¼ 24%, Table 17S, entry 6 in ESI†).
of racemic ethanols (10 mM) with vinyl acetate (3 equiv.) in
The most efficient biocatalysts (see Table 2) for the acetyla-
presence of the previously selected optimal biocatalysts was tion of the racemic alcohols rac-2b,d,i in neat vinyl acetate
Table 2 Lipase mediated O-acylation of rac-2a–i (40 mM) in neat vinyl acetate at 25 ꢀCa
cb,d (%)
b,c
c
Entry
Substrate rac-2a–i
Optimal biocatalyst
Reaction time (h)
eeS (%)
eeP (%)
1
2
rac-2a
rac-2b
All CaL-B preparates
CaL-B 1 TS*
CaL-B Celite
AK 1 BS
CaL-B 1 TS*
CaL-B Celite
All preparates
AK 1 BS
9.5
132
>99
>99
>99
>99
>99
>99
>99
<1
>99
>99
>99
50
82 (ꢃ1.7)
83 (ꢃ2.1)
94 (ꢃ1.)
91 (ꢃ0.9)
96 (ꢃ2.8)
<1
89 (ꢃ1.4)
52 (ꢃ1.2)
75 (ꢃ0.5)
45 (ꢃ0.5)
46 (ꢃ0.5)
49 (ꢃ0.5)
48 (ꢃ0)
49 (ꢃ0.9)
<1
47 (ꢃ0.5)
34 (ꢃ0.5)
43 (ꢃ0)
3
4
rac-2c
rac-2d
136
136
24
144
11
5
6
7
8
rac-2ee
rac-2g
rac-2h
rac-2i
CaL-B Celite
CaL-B 2 TSC
178
40
a
E [ 200. b Experiments were performed in triplicate and standard deviations from average values are given in brackets. c Determined from peak
areas of GC or HPLC chromatograms. Calculated using the formula c ¼ [eeS/(eeS + eeP)]. e No product was detected even aer 6 days.
d
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based on reaction conversion, proved to be the CaL-B preparates
In the case of rac-1-(10-ethyl-10H-phenothiazin-1-yl)ethan-1-
obtained by sol–gel entrapment in ternary silane precursor ol, rac-2e, a sterically hindered secondary alcohol, even sol–gel
systems with or without deposition on Celite (CaL-B 1 TS* and preparates containing lipase A from C. antarctica, previously
CaL-B 2 TSC, with 43–48% conversion), depending on the reported as an efficient biocatalyst32 were inactive (the acetate
substrate structure, but the adsorbed CaL-B lipase was similarly was not detected even aer 6 days, see Table 6S in ESI†).
good with 46–49% conversion. Although the entrapped pre-
The resolution of rac-1-(10-ethyl-10H-phenothiazin-4-yl)
parates have the overall advantage of less enzyme leaking, in ethan-1-ol, rac-2h with CaL-B immobilized preparates in neat
almost anhydrous organic solvents the adsorbed enzyme could vinyl acetate occurred very slowly, but in a selective manner
also exhibit high stability, as it was recently demonstrated for (Table 8S in ESI†). The best results were obtained with CaL-B on
Burkholderia cepacia lipase.37
Celite 545 (c ¼ 34% aer 178 h, E [ 200, entry 7, Table 2).
Lipase from Pseudomonas uorescens (Amano AK), already Based on the solvent screening, performed in the same manner,
reported as efficient,29 demonstrated excellent selectivity for the n-hexane was also selected as optimal solvent since the reaction
EKR of rac-2c in neat vinyl acetate (entry 3 in Table 2); the rate signicantly increased and the maximum 50% conversion
optimal biocatalyst was obtained by sol–gel immobilization in was obtained aer only 27 h (entry 7 in Table 15S of ESI†). As
a binary silane precursor system (AK 1 BS).
already reported,31,32 during the enzymatic reactions and
The most appropriate solvent for the transesterication particularly during the work-up of the reaction mixture, the
reactions of rac-2a–d,i proved to be n-hexane in all cases (E [ instability of the phenothiazinic compounds (ethanol and
200 at c ¼ 46–50% aer 1–28 hours, depending on the substrate, acetate) was observed, mainly for rac-2h.
see Table 3).
It is noticeable that the best performing biocatalysts have
In an effort to nd a general enzymatic route for the shown comparable biocatalytic power with the extensively used
synthesis of enantiomerically enriched phenothiazinic- Novozyme 435 in the transesterication reactions of rac-
ethanols, an enzyme and solvent screening was next per- 2a,b,d,g,i in neat vinyl acetate (Tables 2S, 3S, 5S, 7S and 9S†).
formed on the model substrate rac-2g and the best reaction Even though the acylation reaction of rac-2d in neat vinyl
conditions (lipase preparate and solvent) were applied to the acetate occurred faster with Novozyme 435 (c ¼ 50% aer 16 h,
other phenothiazinic substrates. Accordingly, the enzymatic entry 9, Table 5S†) than with any of the sol–gel CaL-B preparates
kinetic resolution of rac-2g with CaL-B and L-AK immobilized (c ¼ 48% aer 136 h with CaL-B 1 TS*, entry 10, Table 5S†), the
preparates in neat vinyl acetate was rstly investigated. AK 1 enantiomeric excess of the product was smaller (eeP ¼ 97%
BS was the only appropriate enzyme preparate in neat vinyl versus eeP > 99%). In terms of conversion Novozyme 435 proved
acetate (47% conversion aer 11 h, entry 14 in Table 7S in to be more efficient in the acylation reaction of rac-2h in neat
ESI†). When performing the resolution of rac-2g with this vinyl acetate (c ¼ 47% aer 42 h, entry 6, Table 8S†), in
biocatalyst and vinyl acetate (3 equiv.) in the selected solvents, comparison to all the tested sol–gel preparates (c ¼ 14% aer
the maximum conversion was obtained in n-hexane (34% 42 h with CaL-B 1 TS*, entry 3, Table 8S†). It must be taken into
aer 2 h and 50% aer 9 h, entries 1 and 4 in Table 14S consideration that Novozyme 435 is fragile under stirring in
of ESI†). These reaction conditions were next applied to the batch mode, oen resulting in the breakage of the particles and
other phenothiazinic substrates rac-2e,f,h, leading to very loss of enzyme activity. Consequently, this biocatalyst might not
good results in case of rac-2f (50% c and E [ 200 aer be a suitable candidate for repeated use. Biocatalysts obtained
8 h, entry 2 in Table 18S of ESI†), while for rac-2e and rac- through sol–gel entrapment, on the other hand, do not have
2h further investigations were performed (Tables 6S–8S in this disadvantage and have proved in our previous work27,35,38
ESI†).
and in other studies11,15,39 remarkable recycling stability.
Table 3 Lipase mediated acylation of rac-2a–i (10 mM) with vinyl acetate (3 equiv.) in n-hexane at 25 ꢀCa
b,c
c
Entry
Substrate rac-2a–i
rac-2a
Biocatalyst
Reaction time (h)
eeS (%)
eeP (%)
cb,d (%)
1
2
CaL-B 1 BSC
CaL-B Celite
CaL-B 1 TS*
CaL-B Celite
AK 1 BS
CaL-B 1 TS*
CaL-B Celite
AK 1 BS
1
5
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
50
50
rac-2b
92 (ꢃ1.6)
48 (ꢃ0.5)
>99
50
3
4
rac-2c
rac-2d
21
19
2
8
9
86 (ꢃ1.2)
94 (ꢃ2.1)
92 (ꢃ2.5)
>99
46 (ꢃ0.5)
49 (ꢃ0.5)
48 (ꢃ0.8)
50
5
6
7
8
rac-2f
rac-2g
rac-2h
rac-2i
AK 1 BS
CaL-B Celite
CaL-B 2 TSC
>99
50
27
28
99 (ꢃ0.5)
50 (ꢃ0)
95 (ꢃ2.2)
49 (ꢃ0.8)
E [ 200. b Experiments were performed in triplicate and standard deviations from average values are given in brackets. c Determined from peak
a
d
areas of GC or HPLC chromatograms. Calculated using the formula c ¼ [eeS/(eeS + eeP)].
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cycle. Between cycles the catalyst was just washed with anhy-
drous n-hexane three times and thereaer immediately reused.
As presented in Fig. 1A and B, the immobilized lipases were
efficient biocatalysts for the EKR of the benzothiophenic
derivative rac-2a in ten consecutive reaction cycles (Tables 19S
and 20S†). The adsorbed CaL-B was even more effective than the
binary sol–gel preparate containing vinyl pending groups, but
by extending the reaction time to more than one hour the
entrapped biocatalyst was also able to give almost complete
conversion of the substrate with high enantiomer purity.
For the EKR of rac-2c the most active immobilized form of AK
was obtained by immobilization of this lipase in a binary sol–gel
material as well, but containing octyl groups instead of vinyl, to
supply the organic functionality. The recycling results (Fig. 2A)
show a signicant decrease of the conversion in only three
reaction cycles (from 42 to 23% c, Table 21S in the ESI†), while
the enantiopurity of the product remained high. The same
biocatalyst was also subjected to recyclability studies in the
acylation reaction of rac-2g. In this case the conversion started
to drop only aer 5 acylation cycles (Fig. 2B, Table 22S in ESI†).
A rigorous comparative evaluation of this enzyme preparate
Operational stability of the immobilized lipase preparates at
repeated use in batch processes
Long-term catalytic efficiency is a key requirement for any
industrial biocatalyst and immobilization should have positive
impact on the operational stability of the enzyme. The sol–gel
entrapped lipases demonstrated excellent reuse efficiency in the
acylation reaction of 2-octanol, showing the signicant poten-
tial of this technique to provide highly efficient solid-phase
biocatalysts for prolonged exploitation.27
However, the reuse capability of the biocatalyst must be
tested for every individual application, since it depends on the
structure of the substrate, the composition, and characteristics
of the reaction system, as well as on the operational parameters.
For this reason, the reusability of the optimal immobilized
lipase preparates was studied in the enantioselective acylations
of rac-2a,c,d,g,i with vinyl acetate, in n-hexane (Fig. 1–4, Tables
19S–24S in ESI†).
Each reaction was repeated with the same enzyme preparate
up to 10 times and has been allowed to proceed to ꢄ50%
conversion, or as long as the preparate remained active and
enantioselective, before the catalyst was subjected to the next
Fig. 1 Multiple use efficiency of the selected biocatalysts for the EKR of rac-1-(benzo[b]thiophen-2-yl)ethan-1-ol rac-2a (10 mM) with vinyl
acetate (3 equiv.) in n-hexane at 25 ꢀC with: (A) CaL-B 1 BSC and (B) Cal-B on Celite 545 (after 40 min reaction time). Error bars represent
standard deviations from average.
Fig. 2 Multiple use efficiency of AK 1 BS as selected biocatalyst for the EKR of: (A) rac-1-(benzofuran-2-yl)ethan-1-ol rac-2c (10 mM) with vinyl
acetate (3 equiv.) in n-hexane at 25 ꢀC (after 20 hours reaction time) and (B) rac-1-(10-ethyl-10H-phenothiazin-3-yl)ethan-1-ol rac-2g (10 mM)
with vinyl acetate (3 equiv.) in n-hexane at 25 ꢀC (after 8 hours reaction time). Error bars represent standard deviations from average.
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sol–gel matrix and the catalytic efficiency, the presence of bulky
and mid-polar phenyl groups could have a specic inuence on
the structural organization and hydrophobicity of the formed
silica network, resulting in improved performance, as it was
also observed in our previous studies.15,40
The operational stability of a biocatalyst obtained by the
immobilization method involving the formation of a sol pre-
polymer in acid catalysis (Method 2) was investigated for the
EKR of the phenyl-thiazolyl derivative rac-2i.
As results from Fig. 4, this immobilized CaL-B lipase was not
suitable for repeated use, although in the screening study
demonstrated comparable catalytic efficiency and enantiose-
lectivity with the best performing preparates obtained with the
method using NaF as catalyst (Method 1). The most important
drawback was the quick decline of the catalytic performance, as
shown by the low conversion values (falling from 49 to 17% c in
the second cycle, see Table 24S in ESI†). The stability of this type
of sol–gel matrix is probably not appropriate for the employed
reaction conditions.
Fig. 3 Multiple use efficiency of the selected biocatalyst CaL-B 1 TS*
for the EKR of rac-1-(benzofuran-3-yl)ethan-1-ol rac-2d (10 mM) with
vinyl acetate (3 equiv.) in n-hexane at 25 ꢀC (after 20 hours reaction
time). Error bars represent standard deviations from average.
Lipase-mediated acylation of racemic heteroaromatic
ethanols in continuous-ow packed-bed microreactors
The best performing immobilized lipase preparates were also
tested in continuous-ow experiments using packed-bed micro-
reactors. From all CaL-B preparates, CaL-B 1 TS* was selected and
from AK preparates, lipase AK 1 BS was selected. The two
benzofuranyl-ethanols rac-2c,d were chosen as substrates (Fig. 5).
According to preliminary results of experiments performed
in continuous-ow system, under identical reaction conditions,
an increase in the amount of vinyl acetate for the acylation
ꢀ
reaction of rac-2c (catalysed by AK 1 BS, in n-hexane, at 65 C
and 0.5 mL minꢁ1, substrate concentration 10 mM) did not
have a major impact on the conversion (20% c with 0.75 equiv.
of vinyl acetate and 22% c with 3 equiv. of vinyl acetate), while
for the acylation reaction of rac-2d (catalysed by CaL-B 1 TS*, in
Fig. 4 Multiple use efficiency of the selected biocatalyst CaL-B 2 TSC
for the EKR of rac-1-(2-phenyl-thiazol-4-yl)-ethanol rac-2i (10 mM)
with vinyl acetate (3 equiv.) in n-hexane at 25 ꢀC (after 28 hours
reaction time). Error bars represent standard deviations from average.
n-hexane, at 35 C and 0.5 mL minꢁ1, substrate concentration
ꢀ
10 mM) rising the amount of acyl donor led to a signicant
increase in conversion (8% c with 0.75 equiv. of vinyl acetate
and 23% c with 3 equiv. of vinyl acetate). For this reason, in all
further experiments involving rac-2c 0.75 equiv. of vinyl acetate
were used and for rac-2d, 3 equiv. of the same acylating agent
were employed.
performance is not possible, since the time needed to reach the
maximum conversion strongly depends on the substrate struc-
ture (20 h for rac-2c and 8 h for rac-2g). These results conrm
that the recycling capacity of a biocatalyst depends on the
structure of the substrate.
Although rac-2d is a positional isomer of rac-2c, it was
demonstrated to be a substrate with different behaviour in the
EKR process. In this case, CaL-B lipase was the most efficient
biocatalyst (not Amano AK, as for rac-2c) and the best immo-
bilized enzyme showed high efficiency throughout multiple use
(Fig. 3, Table 23S in ESI†).
Firstly, the continuous-ow acylation reaction was investi-
gated at different temperatures (25–65 ꢀC) using a 10 mM
The ternary sol–gel matrix was obtained from tetramethoxy-
silane as building block of the inorganic structure, with phenyl-
trimethoxysilane and methyl-trimethoxysilane (at 4 : 1 relative
molar ratio), supplying the pending organic functional groups
of the hybrid material. The presence of relatively high phenyl
group concentration was benecial for the long-term catalytic
efficiency of this biocatalyst, which did not show notable
decline of conversion and enantioselectivity throughout 10
Fig. 5 Continuous-flow lipase mediated kinetic resolution of racemic
recycles. Although it is difficult to correlate the structure of the benzofuranyl-ethanols rac-2c,d.
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substrate solution at 0.5 mL minꢁ1 ow rate (Fig. 6A). For both the ow rate was gradually decreased from 0.5 to 0.1 mL minꢁ1
,
lipase preparates, an increase in conversion was observed by maintaining the temperature at 65 ꢀC, which led to almost
increasing the temperature, the maximum value being reached complete conversion of the substrates (48% and 50%, Fig. 6B).
at 65 ꢀC. Further increase of the reaction temperature was However it must be taken into consideration that even though
limited by the boiling point of the employed solvent. In the case the conversion increases, the productivity of the continuous-
of CaL-B 1 TS*-mediated acylation of rac-2d, almost a linear ow system can decrease at lower ow rates. This however
increase in conversion with respect to temperature was depends on the magnitude of conversion increase and ow rate
observed, while for AK 1 BS-catalyzed acylation almost a 10% decrease, since the rate of product formation is not a linear
ꢀ
increase in conversion was seen from 45 to 50 C.
function on conversion.
This experiment revealed that the immobilized lipase B from
Furthermore, in an effort to enhance the productivity of the
C. antarctica is more efficient towards rac-2d than is the continuous-ow packed-bed microreactor, different substrate
immobilized lipase from Pseudomonas uorescens towards rac- concentrations were investigated at 65 ꢀC at 0.1 (Fig. 6C) and 0.5
2c, as a two times higher conversion was reached at 65 ꢀC (41% mL minꢁ1 ow rate (Tables 4 and 5).
vs. 20%). The continuous-ow reactions proceeded with excel-
It was noted that for CaL-B 1 TS*-mediated trans-
lent enantioselectivities (E [ 200) throughout the investigated esterication of rac-2d, there was no signicant decline in
ꢀ
temperature domain (25–65 C).
conversion in the range of 10–50 mM substrate concentration at
With the aim to improve the conversion of the enantiose- 0.1 mL minꢁ1
.
lective acylation of the investigated racemic ethanols rac-2c,d,
Even when a 100 mM substrate concentration was employed,
the conversion was still high (44%), proving the efficiency of
this biocatalyst. In the case of the immobilized preparate AK 1
BS, the conversion of the acylation reaction of rac-2c decreased
as the substrate concentration increased and at 100 mM the
conversion was half of the initial value. At higher substrate
concentrations (10–100 mM) the enantiomeric excesses of the
products were maximum (>99%), affording excellent enantio-
selectivities of the continuous-ow acylation reactions.
The maximum productivity achieved by lipase AK 1 BS was
37.4 mmol of product minꢁ1 gꢁ1 at 65 ꢀC, 0.5 mL minꢁ1 ow rate
and 100 mM substrate concentration (Table 4, entry 20), while
the maximum productivity of CaL-B 1 TS* was 157.5 mmol of
product minꢁ1 gꢁ1 at 65 ꢀC, 0.5 mL minꢁ1 ow rate and 50 mM
substrate concentration (Table 5, entry 18).
The long term operational stability of the two selected bio-
catalysts was also studied using a 10 mM substrate concentra-
tion and 0.1 mL minꢁ1 ow rate. The obtained results showed
that the immobilized preparate AK 1 BS maintained 81% of its
initial activity at 65 ꢀC aer 44 h of continuous operation (data
not shown).
At 55 ꢀC the enzyme exhibited remarkable stability as almost
95% of the initial activity was kept aer 100 h. The other bio-
catalyst, CaL-B 1 TS* proved to be even more stable, the results
showing practically no loss of activity aer 100 h at 65 ꢀC
(Fig. 7). The enantioselectivities of both lipase-mediated
continuous-ow reactions were not affected during long term
operation of the biocatalysts.
Another goal was to compare the productivities of the
continuous-ow systems with those obtained in batch mode by
calculating the specic reaction rates (eqn (1) and (2)):
ꢀ
ꢁ
ꢁ
nP
mmol
rbatch
¼
¼
(1)
t ꢂ me min ꢂ g
ꢀ
½Pꢅ ꢂ f mmol
rflow
(2)
me
min ꢂ g
Fig. 6 Effect of temperature (A), flow rate (B) and substrate concen-
tration (C) on the continuous-flow enzymatic acylation of rac-2c with
AK 1 BS ( ) and rac-2d with CaL-B 1 TS*( ) with vinyl acetate (0.75
and 3 equiv., respectively) in n-hexane.
The specic reaction rate of a batch reaction (rbatch) can be
calculated from the amount of product formed (np, mmol), the
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Table 4 Influence of temperature, flow rate and substrate concentration on the continuous-flow kinetic resolution of rac-2c with vinyl acetate
(0.75 equiv.) in n-hexane mediated by AK 1 BS
Flow rate
Residence
Substrate
concentration [mM]
Temperature
[ꢀC]
Productivityd
[mL minꢁ1
]
timea [min]
eeS (%)
[mmol minꢁ1 gꢁ1
]
b,c
Entry
1
2
3
4
5
6
7
8
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.4
0.3
0.2
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.17
0.17
0.17
0.17
0.17
0.17
0.17
0.21
0.28
0.42
0.85
0.17
0.85
0.17
0.85
0.17
0.85
0.17
0.85
0.17
0.85
10
10
10
10
10
10
10
10
10
10
10
20
20
30
30
40
40
50
50
100
100
25
35
45
50
55
60
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
2 (ꢃ0.5)
0.9
2.3
2.3
7.0
7.0
8.4
9.3
8.2
9.0
6.5
4.5
15.9
8.0
22.4
12.3
26.2
16.4
32.7
17.3
37.4
22.4
5 (ꢃ1.2)
5 (ꢃ1.4)
17 (ꢃ0.5)
17 (ꢃ1.7)
22 (ꢃ2.4)
25 (ꢃ0.5)
27 (ꢃ1.2)
47 (ꢃ1.6)
54 (ꢃ2.4)
91 (ꢃ3.1)
21 (ꢃ1.7)
76 (ꢃ1.2)
19 (ꢃ2.1)
78 (ꢃ1.7)
15 (ꢃ1.7)
78 (ꢃ2.1)
16 (ꢃ0.5)
57 (ꢃ2.5)
9 (ꢃ1.2)
9
10
11
12
13
14
15
16
17
18
19
20
21
31 (ꢃ0.5)
volume of liquid inside the packed bed microreactor
a
b
Calculated with the formula RT ¼
½minꢅ. Experiments were performed in triplicate and
flow rate
c
d
standard deviations from average values are given in brackets. Determined from peak areas of HPLC chromatograms. Calculated using the
ꢀ
ꢁ
½Pꢅ ꢂ f mmol
average values of the conversions with the formula rflow
¼
.
me
min ꢂ g
Table 5 Influence of temperature, flow rate and substrate concentration on the continuous-flow kinetic resolution of rac-2d with vinyl acetate
(3 equiv.) in n-hexane mediated by CaL-B 1 TS*
Flow rate
Residence
Substrate
concentration [mM]
Temperature
[ꢀC]
Productivityd
[mL minꢁ1
]
timea [min]
eeS (%)
[mmol minꢁ1 gꢁ1
]
b,c
Entry
1
2
3
4
5
6
7
8
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.4
0.3
0.2
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.31
0.42
0.62
1.25
0.25
1.25
0.25
1.25
0.25
1.25
0.25
1.25
0.25
1.25
10
10
10
10
10
10
10
10
10
10
10
20
20
30
30
40
40
50
50
100
100
25
35
45
50
55
60
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
22 (ꢃ2.1)
30 (ꢃ0.5)
42 (ꢃ2.1)
49 (ꢃ2.4)
58 (ꢃ2.1)
63 (ꢃ0.5)
69 (ꢃ1.7)
72 (ꢃ0.8)
78 (ꢃ2.4)
91 (ꢃ0.9)
>99
72 (ꢃ1.2)
95 (ꢃ2.5)
49 (ꢃ2.2)
>99
30 (ꢃ0.8)
91 (ꢃ0.5)
47 (ꢃ2.5)
95 (ꢃ2.4)
14 (ꢃ1.7)
78 (ꢃ1.7)
17.7
22.6
29.5
32.5
36.4
38.4
40.4
33.1
26.0
18.9
9.8
82.7
19.3
97.4
29.5
90.6
37.8
157.5
48.2
118.1
86.6
9
10
11
12
13
14
15
16
17
18
19
20
21
volume of liquid inside the packed-bed microreactor
a
b
Calculated with the formula RT ¼
½minꢅ. Experiments were performed in triplicate and
flow rate
c
d
standard deviations from average values are given in brackets. Determined from peak areas of HPLC chromatograms. Calculated using the
ꢀ
ꢁ
½Pꢅ ꢂ f mmol
average values of the conversions with the formula rflow
¼
.
me
min ꢂ g
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rac-2a,d), average (5 cycles for rac-2g) or modest (approx. 55%
enzymatic activity recovered aer three cycles for rac-2c, 35%
enzymatic activity recovered aer two cycles for rac-2i).
Furthermore for the resolution of the racemic benzofuranyl-
ethanols (rac-2c,d) the best performing biocatalysts were
studied in continuous-ow mode at different temperatures, ow
rates and substrate concentrations, using the productivity as
criterion. The calculated specic reaction rates under
continuous-ow operation (row) were in both cases higher than
those obtained in batch mode (rbatch), 4.5 and 1.1 mmol minꢁ1 gꢁ1
for AK 1 BS preparate and 9.8 and 6.7 mmol minꢁ1 gꢁ1 for CaL-B 1
TS* preparate, respectively, demonstrating the advantages of this
operating system. Moreover, these biocatalysts showed very good
stability under long term continuous operation at high temper-
Fig. 7 Long term operational stability of immobilized preparates AK 1
BS and CaL-B 1 TS* in the continuous-flow acylation reactions of rac-
2c,d (10 mM) with vinyl acetate (0.75 and 3 equiv., respectively) in n-
hexane at 55 ꢀC and 65 ꢀC, respectively and 0.1 mL minꢁ1 flow rate.
Error bars represent standard deviations from average.
ꢀ
atures (65 C). In conclusion the use of tailored sol–gel immo-
bilized biocatalysts in packed-bed reactors under continuous
ow regime represents a good strategy to perform lipase cata-
lyzed kinetic resolutions of heteroaromatic ethanols and offers
a good background for process development.
reaction time (t, min), and the mass of the applied enzyme
(me, g). Similarly the specic reaction rate of a continuous-ow
reaction (row) can be determined from the concentration of the
product ([P], mmol mLꢁ1), the ow rate (f, mL minꢁ1), and the
mass of the applied enzyme (me, g).
Conflicts of interest
There are no conicts to declare.
A rigorous comparison between these two can be made at the
same degree of conversions, using identical experimental
conditions (substrate and acyl donor concentrations, solvent,
and temperature).41
In the case of AK 1 BS-catalyzed batch and continuous-ow
reactions a signicant difference in the specic reaction rates
values was observed, this biocatalyst affording a 4 times higher
Acknowledgements
This work was supported by a grant of the Romanian National
Authority for Scientic Research, CNDI–UEFISCDI, project
number PN-II-PT-PCCA-2013-4-0734.
productivity under continuous-ow operation (rbatch
¼
Notes and references
1.1 mmol minꢁꢀ1 gꢁ1, row ¼ 4.5 mmol minꢁ1 gꢁ1, at 48%
conversion, 65 C, 10 mM substrate concentration, 0.75 equiv.
of vinyl acetate and 0.1 mL minꢁ1 ow rate). On the other hand,
CaL-B 1 TS* proved to be efficient in both systems, nevertheless,
the specic reaction rate obtained in continuous-ow mode was
almost 1.5 times higher than the one obtained in batch mode
(rbatch ¼ 6.7 mmol minꢁ1 gꢁ1, row ¼ 9.8 mmol minꢁ1 gꢁ1, at 50%
´
1 J. M. Guisan, Immobilization of enzymes and cells, in Methods
in biotechnology, Humana Press, New York, 2006, vol. 22.
2 L. Cao, Carrier-bound immobilized enzymes: principles,
applications and design, Wiley-VCH, Weinheim, 2005.
3 R. A. Sheldon and S. van Pelt, Chem. Soc. Rev., 2013, 42, 6223.
4 C. Mateo, J. M. Palomo, G. Fernandez-Lorente, J. M. Guisan
and R. Fernandez-Lafuente, Enzyme Microb. Technol., 2007,
40, 1451.
ꢀ
conversion, 65 C, 10 mM substrate concentration, 3 equiv. of
vinyl acetate and 0.1 mL minꢁ1 ow rate).
5 R. A. Sheldon, Adv. Synth. Catal., 2007, 349, 1289.
6 U. Hanefeld, L. Gardossi and E. Magner, Chem. Soc. Rev.,
2009, 3, 8.
Conclusion
The possibility to obtain tailored immobilized biocatalysts for
the EKR of some heteroaromatic secondary alcohols through
entrapement in various sol–gel matrix, starting from binary or
ternary precursor mixtures, was demonstrated. Using the
conversion c, enantiomeric excesses eeP and eeS and enantio-
meric ratio E in order to characterize the enzymatic process
efficiency, the optimal conditions (enzyme preparates and
solvent) were found for each tested substrate.
7 O. Barbosa, C. Ortiz, A. Berenguer-Murcia, R. Torres,
R. C. Rodrigues and R. Fernandez-Lafuente, Biotechnol.
Adv., 2015, 33(5), 435.
8 M. L. E. Gutarra, L. S. M. Miranda and R. O. M. A. de Souza,
Enzyme Immobilization for Organic Synthesis, in Organic
Synthesis Using Biocatalysis, ed. A. Goswami and J. D.
Stewart, Academic Press, Amsterdam, 2016, ch. 4, pp. 99–
126.
´
Moreover the operational stability in batch process was
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