Biocatalytic preparation of dichloropropyl acrylates. Application to the synthesis of poly(dichloropropyl acrylates)

Article abstract of DOI:10.1016/j.molcatb.2013.03.005

The synthesis of dichloropropyl acrylates from dichloropropyl dodecanoates through a transesterification process using diverse commercial lipases and whole cells (fungal resting cells) is presented. The synthesis was carried out in a solvent-free media using a conventional batch system and a packed bed reactor (PBR). The effect of water activity on the process depended on the lipase used. The commercial enzyme CALB (Candida antarctica lipase B immobilized onto a macroporous acrylic resin) showed the best performance as a biocatalyst, achieving a yield of 50% and productivity of 7.2 μmol min^-1 g ^-1 in the batch reactor and 33% and 35.8 μmol min^-1 g^-1 in the PBR. Finally, polymeric material was prepared by suspension polymerization of the dichloropropyl acrylates synthesized using PBR. Particles with diameters between 170 and 380 μm were obtained with a yield of 85% after 18 h reaction. AbbreviationsPBRpacked bed reactorCALBCandida antarctica lipase B immobilized onto a macroporous acrylic resin.

Full text of DOI:10.1016/j.molcatb.2013.03.005

Journal of Molecular Catalysis B: Enzymatic 92 (2013) 7–13
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Biocatalytic preparation of dichloropropyl acrylates. Application to the synthesis
of poly(dichloropropyl acrylates)
a
a
b
a
Yara-Varón Edinson , Eras Joli Jordi , Torres Mercè , Balcells Mercè ,
a
a,∗
Villorbina Gemma , Canela-Garayoa Ramon
Department of Chemistry, University of Lleida, Rovira Roure 191, 25198 Lleida, Spain
a
b
Department of Food Science and Technology, University of Lleida, Rovira Roure 191, 25198 Lleida, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of dichloropropyl acrylates from dichloropropyl dodecanoates through a transesterification
process using diverse commercial lipases and whole cells (fungal resting cells) is presented. The synthesis
was carried out in a solvent-free media using a conventional batch system and a packed bed reactor (PBR).
The effect of water activity on the process depended on the lipase used. The commercial enzyme CALB
Received 15 October 2012
Received in revised form 5 February 2013
Accepted 8 March 2013
Available online 15 March 2013
(
Candida antarctica lipase B immobilized onto a macroporous acrylic resin) showed the best performance
−
1
−1
as a biocatalyst, achieving a yield of 50% and productivity of 7.2 ␮mol min
g in the batch reactor
Keywords:
−1
−1
in the PBR. Finally, polymeric material was prepared by suspension
and 33% and 35.8 ␮mol min
polymerization of the dichloropropyl acrylates synthesized using PBR. Particles with diameters between
70 and 380 ␮m were obtained with a yield of 85% after 18 h reaction.
© 2013 Elsevier B.V. All rights reserved.
g
Biocatalytic transesterification
Dichloropropyl acrylates
CALB
Packed bed reactor
Poly(dichloropropyl acrylate)
1
1
. Introduction
Chlorohydrin acrylates are used as a raw material in the prepa-
dichloropropyl dodecanoates can be synthesized from crude glyc-
erol [10]. This process is shown in Fig. 1.
Fossil oil and acylglycerides are sources of glycerol. As a result
of the escalating prices of petroleum and its derivatives in recent
decades, fossil fuels are increasingly being substituted by fuels of
plant origin, such as biodiesel (fatty acid alkyl esters). Glycerol
(1,2,3-propanetriol) is usually the main by-product in the biodiesel
process, accounting for approximately 10% of the total product by
mass [11]. Although glycerol is used in a number of industries, such
as the cosmetic, paint, automotive, food, tobacco, pharmaceutical,
pulp and paper, leather, and textile sectors [12], biodiesel produc-
tion will continue to rise in the coming years. This growth will result
in a large crude glycerol surplus that cannot be absorbed by current
markets for this product [13]. Consequently, the price of glycerol
will fall. Therefore, the development of processes to convert low-
priced glycerol into higher value products is an excellent strategy
to add value to the production of biodiesel.
ration of a range of polymeric [1] and optical [2] materials. They
also have applications in the production of adhesive polymers
with resistance to exposure to X-rays [3], photosensitive resin
[
4], fire-retardant synthetic fibers [5], and hair care products [6],
as well as in imprint lithography [7]. These acrylic esters can
be prepared in the following ways, (i) by esterification of 2,3-
dichloropropanol with acrylic acid in a non-polar aprotic solvent
in the presence of an acid catalyst [2]; (ii) by means of acrylyl
chloride; and (iii) by alcoholysis of ethyl acrylate [8]. In addi-
tion, we recently described the capacity of diverse commercial
lipases and whole cells (fungal resting cells) to synthesize allyl
and dichloropropyl acrylates from allyl dodecanoate and dichloro-
propyl dodecanoates through transesterification in a batch reactor
[
9]. In that study lipases were used as received without water activ-
ity adjustment. Of the biocatalysts tested, CALB was found to be
best followed closely by MmL (lipozyme from Mucor miehei immo-
bilized onto ion-exchange resin). The maximum yield was obtained
using a 1:1 mole ratio of reagents. In turn, allyl dodecanoate and
To increase the value of glycerol, our group has adopted new
approaches based on the transformation of this polyol into halohy-
drin esters. 2-Chloro-1-(chloromethyl)ethyl esters are halohydrin
esters that can be synthesized from glycerol and carboxylic acid
by an esterification-substitution reaction using chlorotrimethyl-
silane (CTMS) [14]. In turn, microwave irradiation allows the use
of higher reaction temperatures that render mixtures of 2-chloro-
Abbreviations: PBR, packed bed reactor; CALB, Candida antarctica lipase B immo-
bilized onto a macroporous acrylic resin.
1
-(chloromethyl)ethyl esters and 2,3-dichloro-1-propyl esters in
∗ Corresponding author. Tel.: +34 973 702843; fax: +34 973238264.
E-mail address: canela@quimica.udl.cat (C.-G. Ramon).
variable ratios [15]. These products can be used as building blocks
for the synthesis of a range of biologically active natural and
1
381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.03.005

8
Y.-V. Edinson et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 7–13
R
R
O
R
O
OH
Catalyst/Cl-
O
O
O
+
R
Biocatalyst
O
O
O
O
O
O
+
+
+
HO
O
OH OH
Cl
Cl
III
Cl
Cl
O
V
Cl
Cl
VI
Cl
Cl
VII
I
II
IV
VIII
R= CH (CH )
-
3
2 10
Fig. 1. Biocatalytic production of dichloropropyl acrylates from glycerol.
synthetic products [16]. Nevertheless, this procedure is not feasible
with acrylic acid and acrylic esters.
the soil of olive (Olea europaea) orchards in Lleida Catalonia (Spain).
The soil was plated on Petri plates containing tributyrin. After five
days the carboxylesterase activity of the tested fungal strains was
determined and A. fumigatus isolated. The fungal strains isolated
were cultured in a synthetic liquid medium as previously described
[23]. Mycelia were harvested from the whole culture broth by fil-
tration using a Buchner funnel and washed with distilled water
followed by acetone. Mycelia were then dried under vacuum for
18 h and ground into a powder. The enzymatic units (U) for each
resting cell were determined beforehand on the basis of the enzy-
matic hydrolysis rate of methyl stearate [9].
Conversely, the enzymatic preparation of chlorohydrin acrylates
using lipases (EC 3.1.1.3) allows reactions to be performed under
mild conditions. The lipases can be used in pure form or in an immo-
bilized form on a support. These conditions prevent the formation
of by-products because the reactions take place at low tempera-
tures, thus greatly reducing the risk of acrylate polymerization. In
addition, enzymes have greater specificity and high selectivity than
conventional chemical catalysts [17].
Transesterification processes can be performed using either a
batch reactor or a packed-bed reactor (PBR), the latter being one
of the most commonly used reactors in biotechnology [18]. A PBR
has several advantages over a batch reactor, including a relatively
short reaction time [19], easy separation of products [20], and reuse
of biocatalyst. Moreover, the ratio between substrate and enzyme
is much lower in a PBR than in a conventional batch reactor, thus
resulting in higher reaction performance [21].
Here, the enzymatic transesterification of ethyl acrylate (V)
with 2-chloro-1-(chloromethyl)ethyl dodecanoate (III) and 2,3-
dichloro-1-propyl dodecanoate (IV) using diverse biocatalysts in
a batch reactor and in a PBR has been studied. The process
was carried out in a solvent-free manner. The PBR allowed the
synthesis of monomers at a sufficient gram scale to prepare a
poly(dichloropropyl acrylate).
2.3. Procedure for the preparation of 2,3-dichloro-1-propyl
dodecanoate (VII)
2
-Chloro-1-(chloromethyl)ethyl dodecanoate (III) was pre-
pared from glycerol (I) and dodecanoic acid (II) following a
previously described method [24]. The isomerization reaction of III
to IV was achieved by microwave irradiation (300 W max, 17 atm
◦
max, 243–247 C) for 1 h in a solvent-free system. The isomerization
process was previously evaluated [15].
2.4. Equilibration of water activity
The water activity (aw) in the experiments was set by inde-
pendently equilibrating isomeric mixtures III–IV, reagent V, and
biocatalyst with aqueous saturated solutions of LiCl (aw = 0.12),
2
. Methods
MgCl (aw = 0.33), K CO (aw = 0.42), Mg(NO ) (aw = 0.54) and NaCl
2
2
3
3 2
2.1. Materials
(aw = 0.75). Separate closed containers were used for each reactant
and biocatalyst [25]. Equilibration was performed at room temper-
ature for at least 48 h. The water activity of the biocatalyst was
measured using an Aqua Lab series 3TE from Decagon Devices Inc.
Candida antarctica lipase B immobilized onto a macroporous
acrylic resin (CALB) from Novozymes A/S, Amano lipase PS-IM
immobilized on diatomaceous earth (PS-IM), lipase immobilized on
Immobead 150 from Rhizopus oryzae (IM-150), lipozyme immobi-
lized from Mucor miehei (MmL) from Novo Nordisk A/S Corp. Amano
lipase PS (Burkhloderia cepacia) (PSL). All lipases were purchased
from Sigma–Aldrich. Dodecanoic acid and ethyl dodecanoate
were purchased from Sigma–Aldrich (Sigma–Aldrich Quimica, S.A.,
Madrid, Spain). Ethyl acrylate and butyl acrylate were from Fluka
(Pullman, WA, USA).
2.5. Enzymatic transesterification in the batch reactor
A 1:1 mixture of V (1 mmol, 100.12 mg) and dichloropropyl
dodecanoates (100:0 or 32:68 III and IV isomeric mixtures, 1 mmol,
311.29 mg) was stirred continuously at 1200 rpm in an orbital
shaker (Eppendorf® Thermomixer Comfort) and at atmospheric
pressure in a reaction vial (1.5 mL) fitted with a PTFE-lined cap.
Reaction vials were used as received. Two sets of experiments were
carried out in a solvent-free system using either 3.3% (10.3 mg) or
15% (46.7 mg) of biocatalyst (commercial lipases and fungal resting
cells) based on the weight of dichloropropyl dodecanoates. Blank
experiments were carried out for each aw studied without biocat-
alysts. Samples were collected at 1 h and 24 h, depending on the
experiment performed. The reaction temperature was chosen on
the basis of the previously described optimum temperature for each
commercial enzyme [9]. For resting cells, the temperature used was
(
Sigma–Aldrich, Madrid, Spain). Hexane and tert-butyl methyl
ether (MTBE) were supplied by J.T. Baker (Quimega, Lleida, Spain).
2
.2. Procedure for obtaining resting cells
The strain of Rhizopus oryzae (RoL) was isolated from plants
of Foeniculum vulgare as endophytic fungi. The plants were col-
lected at The Montsec Natural Park in Catalonia (Spain). Healthy
plant tissues were harvested. The tissues were washed and cut
in pieces (2–3 cm long). All pieces were surface-sterilized by
sequential washes in 0.53% sodium hypochlorite (2 min) and 70%
ethanol (2 min) and rinsed with sterile distilled water. The surface-
sterilized pieces were placed on 2% malt extract agar in Petri dishes.
All plates were incubated at room temperature for a maximum of 2
weeks. Fungi growing out from the plant tissues were transferred to
mycological agar [22]. Aspergillus fumigatus (Af) was isolated from
◦
◦
40 C. Blank assays were conducted at 50 C. Once the experiment
had ended, an aliquot of 10 mg of the crude product was dissolved
in hexane containing an internal standard (typically butyl acrylate).
The resulting solution was analyzed using gas chromatography

Y.-V. Edinson et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 7–13
9
(
GC-FID). Quantification of each compound was achieved using the
Mucor miehei (MmL). A mixture of 0.172 mmol of each dichloro-
propyl ester (independently), 30 mg of biocatalyst and 10 ␮L of
water in 1 mL of MTBE was stirred (1200 rpm) for 24 h at 40 C.
internal standard. Experiments were performed in triplicate.
◦
2.6. Continuous packed-bed reactions
2,3-Dichloro-1-propanol was analyzed by chiral GC in an Agilent
7
890 A series gas chromatograph (Barcelona, Spain) using a CP
The PBR consisted of a glass column (inner diameter: 3 mm; total
Chirasil–DEX CB column (25 m × 0.25 mm diameter, 0.25 ␮m film
length: 100 mm; packed length: 90 mm; inner volume: 0.70 mL)
filled with ca. 240 mg of the corresponding biocatalyst. The glass
column was packed manually with the biocatalyst, which was then
fixed in place using glass wool plugs. All the mixtures were pumped
through the column reactor filled with the corresponding biocat-
alyst at a constant flow rate of 10 ␮L/min (FRX System, Syrris Ltd,
Royston, UK). The column reactor was heated with an FRX Volcano
Column Adaptor (Syrris) and a digitally controlled RCT Basic 40
hotplate (IKA-Werke GmbH & Co., KG, Staufen, Germany) with an
external Pt 100 sensor for optimum control of temperature. A dig-
ital control with an external sensor ensured that the temperature
setting was correctly maintained in the column. Reactions were
performed in a solvent-free system. Two experiments were carried
out. The first experiment consisted on using each described biocat-
alyst, and a mixture of V (2.5 g, 25 mmol) and the III–IV mixture
thickness). Hydrogen (3 ml/min) was used as carrier gas; the injec-
◦
◦
tion system was held at 250 C, and the FID system at 280 C. The
◦
oven temperature was isothermally programmed at 60 C and held
for 120 min
Polymer was vacuum-dried and analyzed using a JASCO FT/IR-
6300 spectrometer, a JASCO NRS-7100 Confocal Laser Raman
spectrometer, and a Varian 400 NMR spectrometer. The C:H:Cl ratio
of each solid was determined by combustion elemental analysis in
a Carlo Erba Instruments EA 1108.
3. Results and discussion
3.1. Synthesis of 2-chloro-1-(chloromethyl)ethyl acrylate
Initially, the transesterification reaction between 2-chloro-1-
(chloromethyl)ethyl dodecanoate (III) and ethyl acrylate in a batch
reactor using several lipases was carried out using a 1:1 mole
ratio of III and V reagents. Five commercially available lipases and
two resting cells at aw = 0.33 at the optimum temperature for each
biocatalyst and either 3.3% or 15% of biocatalyst were used. The
highest yield (22%) was achieved by MmL at 24 h using 15% of
enzyme, followed by CALB (17%) and PSL (15%) under the same con-
ditions (Fig. 2a). The yields obtained using the other biocatalysts
were lower than 15%. Although yields were generally improved
(
7.77 g, 25 mmol). Each experiment was conducted for 24 h, crude
products were recovered and aliquots of 10 mg were dissolved in
hexane containing an internal standard (typically butyl acrylate).
Samples were analyzed as described above. Reaction crude prod-
◦
ucts were vacuum-distilled at 26 C and 0.3 Torr. This distillate was
used for polymerization.
A final experiment was conducted for 72 h, using CALB as
biocatalyst, and a mixture of V (7.5 g, 75 mmol) and the III–IV mix-
ture (23.31 g, 75 mmol). Samples were withdrawn at the indicated
times, dissolved in hexane containing an internal standard (typi-
cally butyl acrylate) and analyzed as described above.
compared to previous study [9] by adjusting a , they were still
w
moderate. This prompted us to study the effect of isomerizing the
1
,3-dichloropropyl radical.
2.7. Preparation of a poly(dichloropropyl acrylate)
3.2. Transesterification process using a regioisomeric mixture of
Suspension polymerization of a distilled mixture of products
III and IV
VI and VII was carried out following the described methodology
26]. Distilled water (5.6 g), poly(vinyl pyrrolidone) (19.8 mg), and
[
Given the results described above, the transesterification using
ascorbic acid (7.89 mg) were added to a reaction vial fitted with
a PTFE-lined cap. The reaction medium was heated to 50 C and
the corresponding chemical mixture of dichloropropyl acrylates
a dichloropropyl dodecanoate mixture with a high content of 2,3-
dichloro-1-propyl dodecanoate (IV) regioisomer was studied. The
yield should be improved considering that the biocatalysts might
react with the primary ester bond much more readily than the
secondary ester bond. The isomerization of III to IV was achieved
by microwave irradiation, obtaining a III:IV regioisomeric ratio of
32:68. This result is in agreement with that described by Escribà
et al. [15]. Fig. 2b shows the results of a set of experiments car-
ried out with this regioisomeric mixture. The maximum yield, 33%,
was achieved by 15% of CALB at 24 h of reaction. This yield increase
might be explained by the fact that although CALB is a highly ver-
satile lipase for esters, it has very limited available space in the
active site pocket compared to other lipases [27]. These features
may explain why CALB shows higher activity when the regioisomer
IV is present in the reaction mixture. Similar behavior was observed
for MmL. Now yield was 30% higher than when the transesterifi-
cation was carried out using MmL and the regioisomer III. MmL
is considered a 1,3-specific lipase [28], which might explain the
yield increase favored by the formation of product VII. The resting
RoL cells showed the best performance, with 15.8% of yield using
15% of biocatalyst. The other biocatalysts showed yields lower than
15% in all cases. Consequently, all subsequent experiments were
conducted at 24 h using 15% of biocatalyst.
◦
(
620 mg) and benzoyl peroxide (BPO) (3.41 mg), used as initiator,
were added under vigorous magnetic stirring (900 rpm). The sys-
◦
tem was allowed to react for 18 h at 70 C. The solid formed were
recovered and washed until no monomer was detected in the wash-
ing solution.
2.8. Analysis
The progress of each reaction was determined by gas
chromatography (GC) using an Agilent HP6890 series gas chro-
matograph (Barcelona, Spain) coupled to a flame ionization
detector (FID). The analytical column was a 30 m × 0.25 mm fused
silica capillary coated with a 0.20 ␮m film of poly(80% bis-
cyanopropyl 20% cyanopropylphenyl siloxane) (SP-2330; Supelco,
◦
Madrid, Spain). The temperature program used was 40 C for 5 min,
◦
followed by an increase of 20 C/min until reaching the final tem-
◦
perature of 225 C, which was then held for 3 min A 1:20 split
injection ratio was used. Hydrogen was used as the carrier gas at
a constant pressure of 620 kPa The injection volume was 1 ␮L. The
◦
◦
injection system was held at 250 C and the FID system at 280 C.
Quantification was performed by a conventional internal standard
method using the corresponding acrylic ester standards.
3.3. Effect of aw on catalytic activity
The enantioselectivity of enzymes was determined through
total hydrolysis of 2,3-dichloro-1-propyl dodecanoate and 2,3-
dichloro-1-propyl acrylate using lipozyme immobilized from
In an attempt to improve the yield of dichloropropyl acrylates,
we evaluated the influence of several water activity values on the

1
0
Y.-V. Edinson et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 7–13
Fig. 3. Yield of dichloropropyl acrylates obtained using diverse commercial
enzymes and resting cells in the batch reactor. Reaction conditions: reaction time
4 h, lipases at a concentration of 15% based on the weight of dichloropropyl dode-
2
canoates. aw: LiCl (0.11), MgCl2 (0.33), K2CO3 (0.42), Mg(NO3)2 (0.54).
values of the substrate. Most lipases have their active center
secluded from the medium by a polypeptide chain called lid or
flap [32]. These lipases follow a well-known mechanism so-called
interfacial activation. This phenomenon occurs when, the lipase
becomes adsorbed on a hydrophobic surface and the lid moves. This
process allows the interaction between reagents and the active cen-
ter (open form). CALB has a very small and simple lid [33]. The active
center is not fully isolated and the enzyme does not show interfacial
activation [34]. In contrast, MmL has a great lid and its interfa-
cial activation is very well known [35]. Consequently, substrates
might easily reach the active center into CALB than into MML. PS-IM
showed the highest yield (18%) at aw = 0.42 while IM-150, PSL and
RoL lipases transformed ethyl acrylate to dichloropropyl acrylate in
a greater proportion at aw = 0.33. These results could be attributed
to each enzyme displaying its highest activity at a different water
activity value [29]. Moreover, mass transfer limitations may also
occur as a consequence of the aggregation of the biocatalysts at
high water activity values [36].
Fig. 2. Yield of dichloropropyl acrylates using lipases at concentrations of
A = 3.3% and B = 15% based on the weight of dichloropropyl dodecanoates. The
reaction was carried out in batch for 1 h and 24 h. aw = 0.33 (MgCl2). (a) 2-
Chloro-1(chloromethyl)ethyl acrylate, (b) mixture of the two regioisomers of
dichloropropyl acrylate.
reaction. Water activity (aw) is one of the most important factors
affecting enzyme activity. Given that excess water favors hydroly-
sis over condensation reactions, water content may affect reaction
rate, yield and enzyme stability [22]. Fig. 3 shows the influence
of aw on the yield of dichloropropyl acrylates. No transesterifica-
tion was observed in the blank assays (data not shown). Again,
CALB showed the best performance. Maximum yield rose from
3.4. Enzymatic transesterification in a PBR
In an attempt to improve the reaction performance, we per-
formed a new set of experiments using a PBR. For this purpose, we
used the enzymes that showed the highest activities in the batch
reactor. Transesterification studies were started using a 1:1 mole
ratio of ethyl acrylate:dichloropropyl dodecanoates, 240 mg of bio-
catalyst (3.3%), the optimal water activity for each enzyme, and the
minimum flow rate allowed for our system (10 ␮L/min). We had
previously determined in an equivalent PBR that transesterifica-
tion with several acrylates reached the highest yields at this flow
rate [26].
The biocatalysts showed the same behavior as in the batch reac-
tor (Fig. 4). Although CALB was again the best lipase in terms of
yield (33%, aw = 0.54), followed by MmL (21%, aw = 0.42), PS-IM
(7%, aw = 0.42) and RoL (6.2%, aw = 0.33), yields were moderately
lower those obtained in the batch reactor. These results may be
attributable to the fact that the amount of biocatalysts in the batch
reactor was five-fold that used in the PBR. Moreover, to evaluate the
extent of enzyme deactivation in the PBR, a 72 h experiment was
carried out using CALB. Fig. 5 shows that yield remained around 32%
for 30 h reaction time. However, the yield decreased to 25% after
54 h and 15% after 72 h. A quadratic polynomial adjustment was
3
3 to 50%. Indeed, this lipase showed the highest yield at all the
water activity values tested. Thus, 50, 47, 33 and 25% yields were
obtained for aw = 0.54, 0.42, 0.33 and 0.11 respectively. The behav-
ior of CALB at various aw values showed that the catalytic activity
and stability of enzymes are markedly influenced by hydration
levels [29]. CALB shows its highest activity at aw = 0.54. Neverthe-
less, we checked CALB activity at aw = 0.75 and observed a clear
decrease in yield (data not shown). In contrast, the dependency
of the yield on water activity for MmL followed a classical bell-
shaped curve in the range of water activity values assayed initially.
These results are in agreement with those described by Wehtje et al.
[
30]. Karra-Chaabouni et al. [31] proposed that this dependency is
due to the simultaneous occurrence of two opposite phenomena:
the first ascending part of the curve (22% yield, aw = 0.33) reflects
the increasing hydration of the enzyme up to its optimum value
(
(
38% yield, aw = 0.42) while the descending part at high aw values
28% yield, aw = 0.54) comes from apparent variations of the KM

Y.-V. Edinson et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 7–13
11
Fig. 4. Yield of dichloropropyl acrylates obtained using a PBR. Reaction conditions:
Fig. 6. Mixture of regioisomers: reagents (III and IV) and products (VI and VII).
continuous flow rate at 10 ␮L/min, 1:1 mole ratio ethyl acrylate:regioisomeric mix-
ture of dichloropropyl dodecanoates, lipases at a concentration of 3.3% based on the
weight of dichloropropyl dodecanoates. aw: MgCl2 (0.33), K2CO3 (0.42), Mg(NO3)2
(
0.54).
3.5. Catalytic activity in a batch reactor versus a PBR
Productivity of the reactions using the so-called specific reaction
rate (r) was determined in order to compare continuous flow and
batch reactions. This parameter indicates the amount of product
that can be formed in one minute by 1 g of enzyme [38]. The produc-
tivity of a given enzyme was higher in the continuous-flow system
than in the corresponding batch reaction (Table 1). This result is in
agreement with that described by Boros et al. [39]. CALB showed
an r in PBR (rPBR) about five-fold higher than in the batch reactor
(rbatch). MmL showed a rPBR more than three-fold higher than rbatch.
For a comparison between the productivity of the two systems, the
same degree of conversion should be considered [40]; however,
given that yields were higher in the batch reactor than in the PBR,
the productivity for the latter might be even higher if conversion
were the same.
Fig. 5. Effect of the reaction time on the yield of dichloropropyl acrylate prepared
using a PBR. Reaction conditions: continuous flow rate at 10 ␮L/min, 1:1 mole ratio
ethyl acrylate:regioisomeric mixture of dichloropropyl dodecanoates, CALB (3.3%
based on the weight of dichloropropyl dodecanoates). aw: Mg(NO3)2 (0.54).
3.6. Regio and enantio selectivity of the biocatalysts
Enzymes can catalyze a variety of reactions with high specificity.
achieved by the minimum square method. Indeed, this observa-
tion suggests that there is an enzyme inactivation, a limited access
of the substrate to the enzyme caused by blockage with product, or
both [37].
Furthermore, regioselective synthesis is amenable using enzymatic
approaches. Therefore, we studied the transformation capacity of
regioisomers (Fig. 6). Table 2 shows the ratio of regioisomers VI and
VII achieved by diverse lipases. In all cases VII was formed in higher
Table 1
−
1
−1
g ). A 1:1 mole ratio ethyl acrylate:regioisomeric mixture of dichloropropyl
Effects of biocatalyst and reactor system on the yield and specific reaction rate (r = ␮mol min
dodecanoates was used in all experiments.
◦
−1 −1
)
Biocatalyst
Biocatalyst support
Specific activity (U g of
protein × 10
T ( C)
Yield (%)
Batcha
r (␮mol min
g
−
1
3)
PBRb
rbatch
rPBR
c
c
CALB
MmL
PS-IM
IM-150
PSL
Acrylic resin
111
0.943
0.143
0.367
34.5
50
50
50
40
50
40
40
50 ± 1.2
38 ± 1.1
18 ± 1.4
13 ± 0.7
11 ± 0.9
14 ± 1.1
14 ± 0.6
33 ± 0.9
7.2 ± 0.5
4.3 ± 0.4
2.2 ± 0.6
1.5 ± 0.3
1.3 ± 0.2
1.9 ± 0.4
1.6 ± 0.5
35.8 ± 1.3
d
d
e
e
e
d
d
d
Ion-exchange resin
Diatomaceous earth
Immobead 150
Powder, free enzyme
Resting cells
21 ± 1.2
13.9 ± 1.1
7 ± 0.8
9.1 ± 0.6
–
–
–
–
e
RoL
Af
6340
175
6 ± 1.1
8.2 ± 0.8
Resting cells
–
–
a
Batch reactions at 24 h (1000 rpm, lipases at a concentration of 15% based on the weight of dichloropropyl dodecanoates).
Flow reactions in the PBR (flow = 10 ␮L/min, samples taken 60 min after starting, column reactor filled with lipases at a concentration of 3.3% based on the weight of
b
dichloropropyl dodecanoates).
c
aw = 0.54.
aw = 0.42.
aw = 0.33.
d
e

1
2
Y.-V. Edinson et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 7–13
Table 2
Yield, product ratio of each regioisomer of dichloropropyl acrylate, and standard deviations obtained in the two reactor systems.
Biocatalyst
Batch
PBR
Yield (%)
VI (%)
VII (%)
Yield (%)
VI (%)
VII (%)
a
a
CALB
MmL
PS-IM
IM-150
PSL
50 ± 1.2
38 ± 1.1
18 ± 1.4
13 ± 0.7
11 ± 0.9
14 ± 1.1
14 ± 0.6
19 ± 0.6
29 ± 1.1
39 ± 0.7
25 ± 1.3
34 ± 1.0
39 ± 0.8
21 ± 1.2
81 ± 1.2
71 ± 1.3
61 ± 0.8
75 ± 0.9
66 ± 1.3
61 ± 1.5
79 ± 0.7
33 ± 0.9
20 ± 0.8
80 ± 1.6
b
b
b
21 ± 1.2
35 ± 1.3
65 ± 1.3
b
7 ± 0.8
36 ± 1.4
64 ± 0.9
c
–
–
–
–
–
–
c
c
c
RoL
Af
6 ± 1.1
42 ± 1.2
58 ± 1.2
b
–
–
–
a
aw = 0.54.
aw = 0.42.
aw = 0.33.
b
c
yields in the batch reactor and in the PBR. Although CALB, PS-IM and
PSL are non-specific lipases [41], these biocatalysts showed high
formation of VII. Nonetheless, CALB was the best enzyme in terms
of activity and regioselectivity. When MmL an 1,3-specific lipase
[
28] was used, a ratio of 29:71 of VI:VII products was obtained
in spite of using a 32:68 ratio of III:IV reagents. The activity of Af
resting cells was lower than that of CALB; however, the formation
of regioisomer VII was similar to this lipase.
On the basis of the formation of the chiral 2,3-dichloro-1-
propyl acrylate, we evaluated the enantioselective capacity of
diverse enzymes during the transesterification reaction. Our ini-
tially attempts to separate the enantiomer esters by chiral gas
chromatography were unsuccessful. Consequently, an indirect
approach was attempted. The hydrolysis of racemic mixtures of
reagent IV and product VII was performed using MmL. Hydroly-
sis of IV and VII was carried out to corroborate that this lipozyme
does not show enantiopreference during hydrolysis reactions. Once
this was confirmed, reagent IV and product VII recovered from the
diverse enzymatic studies were hydrolyzed using the same proce-
dure described above. The results showed that racemic mixtures of
2
,3-dichloro-1-propanols were obtained in all the transesterifica-
tions carried out with the biocatalysts, thereby indicating the lack
of stereoselectivity during the transesterification reactions.
Fig. 7. (a) 1H NMR spectra of initial mixture of dichloropropyl acrylate regioisomers;
b) 1H NMR spectra of poly(dichloropropyl acrylate).
(
3.7. Preparation of poly(dichloropropyl acrylate)
Crude reaction mixtures obtained using the PBR were distilled
and the liquid recovered was analyzed by GC-FID and 1H NMR.
Using CALB as a biocatalyst, we prepared 3.6 g of a mixture of
regioisomers VI:VII (2:8) (Fig. 7a). A typical radical catalyzing poly-
merization method was followed using this mixture. This process
allowed the preparation of solid particles with a wide range of
diameters (170–380 ␮m). The yield was 85% after 18 h of poly-
merization. Fig. 7b shows the 1H NMR spectra of the polymer
obtained. NMR signals at ı 3.7, 4.4 and 5.1 confirm the presence
of the dichloropropyl ester in the polymer.
Fig. 8 shows the FT-IR spectra (a) and Raman spectra (b)
of the polymer obtained after 18 h of reaction. Characteristic IR
bands corresponding to the presence of non-conjugated carboxylic
−1
−1
Fig. 8. FT-IR spectra (a) and Raman spectra (b) of poly(dichloropropyl acrylate).
esters (1732 cm , C O stretching; 1175–1160 and 1046 cm
,
−
1
C
O stretching), alkyl saturated chains (2960 cm , C H stretch-
−1
ing), and aliphatic chloro bond (740 cm C–Cl stretching) can be
observed. The presence of these functional groups was also con-
firmed by the Raman spectra (1740 cm , C O stretching; 1172 and
4. Conclusions
−1
This study shows that the biocatalytic transesterification of
dichloropropyl acrylates can be carried out with commercial
enzymes and fungal resting cells in batch reactor and a PBR. The
biocatalysts showed distinct behaviors in response to water activ-
ity. CALB was the best biocatalyst, achieving its highest activity at
aw = 0.54. CALB can be used in a PBR system up to 30 h without
a yield decrease. The resting cells with lipase-like activity were
−
1
−
1
9
82 cm , C O stretching; 2982–2966 cm and aliphatic chloro
−
1
−1
C Cl stretching; 300 cm ı C C aliphatic chains).
bond 744 cm
The elemental analysis of the polymer revealed an empirical
formula of C24H33Cl O (Elem. Anal. calculated for C24H33Cl O :
8
8
8
8
C, 39.32; H, 4.54; Cl, 38.69; O, 17.46. Found: C, 39.65; H, 4.50; Cl,
8.47; O, 17.38).
3

Y.-V. Edinson et al. / Journal of Molecular Catalysis B: Enzymatic 92 (2013) 7–13
13
found to catalyze the reaction, but always with low to moder-
ate yields. Finally, a water-dispersive polymerization procedure
was carried out to prepared polymeric material. The particles of
poly(dichloropropyl acrylate) achieved reveal that the proposed
process starting from a renewable resource as glycerol can be used
for polymer preparation purposes.
[15] M. Escriba, J. Eras, M. Duran, S. Simon, C. Butchosa, G. Villorbina, M. Balcells, R.
Canela, Tetrahedron 65 (2009) 10370–10376.
[
16] C. Solarte, M. Escriba, J. Eras, G. Villorbina, R. Canela, M. Balcells, Molecules 16
2011) 2065–2074.
(
[17] P. Srimhan, K. Kongnum, S. Taweerodjanakarn, T. Hongpattarakere, Enzyme
Microb. Technol. 48 (2011) 293–298.
[
[
[
18] I. Itabaiana Jr., L.S. de Mariz e Miranda, R.O.M.A. de Souza, J. Mol. Catal. B: Enzym.
5–86 (2013) 1–9.
19] T. Zhao, B.H. Kim, S.I. Hong, S.W. Yoon, C.-T. Kim, Y. Kim, I.-H. Kim, J. Food Sci.
7 (2012) C267–C271.
20] L. Babich, A.F. Hartog, M.A. van der Horst, R. Wever, Chem. Eur. J. 18 (2012)
604–6609.
8
7
Acknowledgements
6
This work was supported in part by a Grant-in-Aid for the
Secretaría de Estado de Política Científica y Tecnológica of the Span-
ish Ministry of Education and Culture (contract grant number:
CTQ2009-14699-C02-01). The authors are grateful to the Vicer-
rectorat de Recerca de l’Universitat de Lleida for the UdL grant of
Edinson Yara Varón. We also thank Tania Yates for the proofreading
of the manuscript.
[21] S.F.A. Halim, A.H. Kamaruddin, W.J.N. Fernando, Bioresour. Technol. 100 (2009)
710–716.
[
22] M. Torres, M.M. Dolcet, N. Sala, R. Canela, J. Agric. Food Chem. 51 (2003)
328–3333.
3
[23] M. Torres, E. Barbosa, V. Loscos, R. Canela, Biotechnol. Lett. 22 (2000)
1265–1268.
[
24] J. Eras, M. Escriba, G. Villorbina, M. Oromi-Farrus, M. Balcells, R. Canela, Tetra-
hedron 65 (2009) 4866–4870.
[
[
25] M. Nordblad, P. Adlercreutz, Biotechnol. Bioeng. 99 (2008) 1518–1524.
26] E. Yara-Varón, J. Eras Joli, M. Balcells, M. Torres, R. Canela-Garayoa, RSC Adv. 2
(
2012) 9230.
References
[
[
27] A. Idris, A. Bukhari, Biotechnol. Adv. 30 (2012) 550–563.
28] P.K. Pal, D.K. Bhattacharyya, S. Ghosh, JAOCS 78 (2001) 31–36.
[
1] B. Gruning, G. Hills, W. Josten, D. Schaefer, S. Silber, C. Weitemeyer, US6268521,
[29] G.V. Chowdary, S.G. Prapulla, Process Biochem. 38 (2002) 393–397.
[30] E. Wehtje, P. Adlercreutz, Biotechnol. Lett. 19 (1997) 537–540.
[31] M. Karra-Chaabouni, S. Pulvin, D. Thomas, D. Touraud, W. Kunz, Biotechnol.
Lett. 24 (2002) 1951–1955.
[32] Y. Bourne, C. Martinez, B. Kerfelec, D. Lombardo, C. Chapus, C. Cambillau, J. Mol.
Biol. 238 (1994) 709–732.
2
001.
[
[
[
[
[
2] H. Kazuhiko, JP1301648, 1989 1–5.
3] J.M. Moran, G.N. Taylor, J. Vac. Sci. Technol. 16 (1979) 2014–2019.
4] S. Arimatsu, K. Konishi, Y. Ichinose, T. Hase, US5397675, 1995.
5] K. Takeya, H. Suzuki, T. Ichimaru, US4022750, 1977.
6] D.G. Anderson, A. Nashat, M.J. DeRosa, D.T. Puerta, R.P. McLaughlin, B.S. Akcasu,
S.A. Williams, M.R. Ramirez, US20100284953, 2010.
[33] M. Skjøt, L. De Maria, R. Chatterjee, A. Svendsen, S.A. Patkar, P.R. Østergaard, J.
Brask, ChemBioChem 10 (2009) 520–527.
[
7] S.F. Wuister, J.F. Dijksman, Y.W. Kruijt-Stegeman, I. Schram, US2011/0266255,
[34] R.D. Schmid, R. Verger, Angew. Chem. Int. Ed. 37 (1998) 1608–1633.
[35] R.C. Rodrigues, R. Fernandez-Lafuente, J. Mol. Catal. B: Enzym. 62 (2010)
197–212.
[36] J.C. Jeong, S.B. Lee, Biotechnol. Tech. 11 (1997) 853–858.
[37] L.L. Woodcock, C. Wiles, G.M. Greenway, P. Watts, A. Wells, S. Eyley, Biocatal.
Biotransform. 26 (2008) 501–507.
2
011.
[
[
8] C.E. Rehberg, M.B. Dixon, W.A. Faucette, J. Am. Chem. Soc. 72 (1950) 5199–5200.
9] E. Yara-Varón, J. Eras Joli, M. Torres, N. Sala, G. Villorbina, J.J. Méndez, R. Canela-
Garayoa, Catal. Today (2012), doi:10.1016/j.cattod.2012.02. 2012 055.
[
10] M. Escribà, J. Eras, G. Villorbina, M. Balcells, C. Blanch, N. Barniol, R. Canela,
Waste Biomass Valor. 2 (2011) 285–290.
[38] E.J. Tomotani, M. Vitolo, J. Food Eng. 80 (2007) 662–667.
[39] Z. Boros, P. Falus, M. Márkus, D. Weiser, M. Oláh, G. Hornyánszky, J. Nagy, L.
Poppe, J. Mol. Catal. B: Enzym. 85–86 (2013) 119–125.
[40] C. Csajagi, G. Szatzker, E.R. Toke, L. Uerge, F. Darvas, L. Poppe, Tetrahedron
Asymmetry 19 (2008) 237–246.
[
[
[
11] S. Hu, X. Luo, C. Wan, Y. Li, J. Agric. Food Chem. 60 (2012) 5915–5921.
12] G.P. da Silva, M. Mack, J. Contiero, Biotechnol. Adv. 27 (2009) 30–39.
13] A. Vlysidis, M. Binns, C. Webb, C. Theodoropoulos, Biochem. Eng. J. 58–59 (2011)
1
–11.
[14] J. Eras, J.J. Méndez, M. Balcells, R. Canela, J. Org. Chem. 67 (2002) 8631–8634.
[41] E. Hernandez-Martin, C. Otero, Bioresour. Technol. 99 (2008) 277–286.