Enzyme-catalyzed synthesis of aliphatic-aromatic oligoamides

Article abstract of DOI:10.1021/bm400243a

Enzymatically catalyzed polycondensation of p-xylylenediamine and diethyl sebacate resulted in oligo(p-xylylene sebacamide) with high melting temperatures (223-230 C) and the enzymatic polycondensation of dimethyl terephthalate and 1,8-diaminooctane leads to oligo(octamethylene terephthalamide) with two melting temperatures at 186 and 218 C. No oligoamides, but products 1 and 2, were formed from the enzymatic reaction of dimethyl terephthalate and p-xylylenediamine. All reactions were catalyzed by CAL-B, icutinase, or CLEA cutinase. All reactions catalyzed by CAL-B show higher conversion than reactions catalyzed by icutinase or CLEA cutinase. The highest DP_max of 15 was achieved in a one-step and two-step synthesis of oligo(p-xylylene sebacamide) catalyzed by CLEA cutinase.

Full text of DOI:10.1021/bm400243a

Article
pubs.acs.org/Biomac
Enzyme-Catalyzed Synthesis of Aliphatic−Aromatic Oligoamides
E. Stavila, G. O. R. Alberda van Ekenstein, and K. Loos*
Department of Polymer Chemistry, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG
Groningen, The Netherlands
S
* Supporting Information
ABSTRACT: Enzymatically catalyzed polycondensation of p-
xylylenediamine and diethyl sebacate resulted in oligo(p-
xylylene sebacamide) with high melting temperatures (223−
230 °C) and the enzymatic polycondensation of dimethyl
terephthalate and 1,8-diaminooctane leads to oligo-
(octamethylene terephthalamide) with two melting temper-
atures at 186 and 218 °C. No oligoamides, but products 1 and
2, were formed from the enzymatic reaction of dimethyl
terephthalate and p-xylylenediamine. All reactions were
catalyzed by CAL-B, icutinase, or CLEA cutinase. All reactions
catalyzed by CAL-B show higher conversion than reactions catalyzed by icutinase or CLEA cutinase. The highest DPmax of 15 was
achieved in a one-step and two-step synthesis of oligo(p-xylylene sebacamide) catalyzed by CLEA cutinase.
antarctica lipase B (CAL-B) immobilized on an acrylic resin
(commercially known as N435) was reported.15 In contrast to
this the enzymatic polymerization of aliphatic−aromatic
polyesters has been frequently using lipase as the catalyst.15,22,23
In our previous study, we reported that the cross-linked
enzyme aggregate (CLEA) from Fusarium solani pisi cutinase
has a good synthetic activity for the enzymatic synthesis of
nylons.7 Therefore, in our present research, we used cutinase
for the synthesis of aliphatic−aromatic oligoamides; we
determined the catalytic activity of the cutinase toward
aromatic monomers and compared its activity with CAL-B.
We present the enzymatic synthesis of aliphatic−aromatic
oligoamides by polycondensation of diethyl sebacate and p-
xylylenediamine; and dimethyl terephthalate and 1,8-diami-
nooctane. Additionally, the enzymatic reaction of dimethyl
terephthalate and p-xylylenediamine is reported in this paper.
All reactions are catalyzed by CAL-B, immobilized cutinase on
Lewatit (icutinase), or CLEA cutinase, as shown in the Scheme
1.
INTRODUCTION
■
Polyamides are widely used polymers in daily and industrial
applications due to their high mechanical strength and good
thermal resistance.1 The repeating units in polyamides can be
aliphatic, aromatic, or a combination of aliphatic and aromatic.
Aliphatic polyamides, commercially known as nylons, which are
frequently used as fiber materials, are synthesized by polymer-
ization of aliphatic monomers. Polyamides that are synthesized
from aromatic monomers are called aramids and have better
mechanical and thermal properties than their aliphatic analogs.2
Commercial aramids, such as poly(p-phenylene terephthala-
mide) (PPPT) and poly(m-phenylene isophthalamide) (PMPI)
are used as electrical insulations, bullet-proof body armor,
industrial fillers, and so on.3 Certain polyamides are synthesized
from a combination of aliphatic and aromatic monomers. They
are known as aliphatic−aromatic polyamides and mostly have
better solubility than aramids and better mechanical and
thermal properties than nylons.
The industrial synthesis of most polyamides is carried out by
a melting process. However, for the synthesis of aramid and
aliphatic−aromatic polyamides, the melting process is very
difficult to perform due to the high melting temperatures and
relatively low decomposition temperatures of the polyamides
from this group.3,4 For an alternative synthetic method
enzymatic polymerizations are a very good choice; not only
because of their mild conditions but also due to their
environmentally friendliness.5−9
EXPERIMENTAL SECTION
■
Materials. Fusarium solani pisi cutinase (Novozym 51032) was
kindly provided by Novozymes, Denmark. Lewatit OC VOC 1600 was
donated by Lanxess, Belgium. Ethanol, formic acid, and calcium
hydride were purchased from Merck. Diethyl sebacate, p-xylylenedi-
amine, 1,1,1,3,3,3-hexafluoro-2-isopropanol, sodium trifluoroacetate,
Lipase acrylic resin from Candida antarctica lipase B (CAL-B
commercially available as N435), molecular sieves 4 Å, and
trifluoroacetic acid-d (TFA-d) were purchased from Sigma-Aldrich.
1,8-Diaminooctane, dimethyl terephthalate, and 2-(4-hydroxy-
phenylazo)benzoic acid (HABA) were purchased from Fluka. Except
Lipases are the most used enzymes in the synthesis of
polyamides and polypeptides, due to their broad substrate
specificity.6 Lipases are used not only in polymerizations, but
also in transamidations10,11 and aminolysis reactions.12,13 Most
reported enzymatically synthesized polyamides are ali-
phatic.6−8,14−21 Best to our knowledge, so far only the synthesis
of silicone aromatic polyamides (SAPAs) using Candida
Received: February 14, 2013
Revised: March 29, 2013
Published: April 1, 2013
© 2013 American Chemical Society
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icutinase, or CLEA cutinase), diester (2.5 mmol), diamine (2.5 mmol),
and 0.5 g of dried molecular sieves. The first step of the reaction was
carried out at 90 °C at normal pressure for 2 h and followed by
decreasing the pressure to 500 mmHg for the next 22 h. The second
step was carried out by decreasing the pressure to 100 mmHg for 24 h.
Then, an aliquot from the residue was analyzed by 1H NMR in TFA-d
for conversion determination. Subsequently, filtration and washing
with CH3OH were performed to separate diphenyl ether from the
reaction mixtures. The next steps, purification and isolation of the
products are identical to the one-step procedure above.
Scheme 1. Enzymatic Reaction between Aliphatic or
Aromatic Diamine and Diester
Oligo(p-xylylene sebacamide) 1H NMR (400 MHz, TFA-d): δ 8.05
(s, 4H, CH), 7.48 (m, 2H, CH), 7.39 (d, J = 8.27 Hz, 2H, CH), 7.32
(d, J = 8.13 Hz, 2H, CH), 7.26 (s, 4H, CH), 4.60 (d, J = 7.59 Hz, 4H,
CH2), 4.29 (s, 2H, CH2), 4.17 (m, 2H, CH2), 2.64 (s, 4H, CH2), 2.39
(s, 2H, CH2), 1.68 (s, 4H, CH2), 1.26 (m, 11H, CH2 and CH3).
1
Oligo(octamethylene terephthalamide) H NMR (400 MHz, TFA-
d): δ 8.18 (d, J = 9.35 Hz, 2H, CH), 7.84 (d, J = 8.88 Hz, 2H, CH),
4.04 (s, 3H, CH3), 3.61 (t, J = 7.83 Hz, 4H, CH2), 1.74 (s, 4H, CH2),
1.41 (s, 8H, CH2).
Dimethyl 4,4′-(((1,4-phenylenebis(methylene))bis(azanediyl))bis-
(carbonyl))dibenzoate (product 1) and 4-((4-((4-(methoxycarbonyl)-
benzamido)methyl)benzyl)carbamoyl)benzoic acid (product 2). 1H
NMR (400 MHz, TFA-d): δ 8.24 (m, 2H, CH), 8.17 (d, J = 8.34 Hz,
4H, CH), 7.88 (d, J = 8.51 Hz, 4H, CH), 7.36 (s, 4H, CH), 4.74 (s,
4H, CH2), 4.03 (s, 6H, CH3). MS (ESI) Product 1: m/z = 461.17 [M
+ H]+, 483.15 [M + Na]+, and 499.12 [M + K]+; Product 2: m/z =
447.15 [M + H]+.
Control Reactions for Enzymatic Synthesis. As control
reactions, one-step and two-step synthesis were performed without
the addition of enzyme. The control reactions were carried out using
the same equimolar amounts of diester and diamine as in the one-step
and two-step reactions.
Instrumental Methods. Attenuated total reflection-Fourier trans-
form infrared (ATR FT-IR) measurements were carried out on a
Bruker IFS88 FT-IR spectrometer. 1H NMR measurements were
performed on a 400 MHz Varian VXR apparatus, TFA-d as solvent.
The signals were referenced to tetramethylsilane (δ = 0.00 ppm).
MALDI-ToF MS measurements were performed on a Biosystems
Voyager-DE PRO spectrometer, in positive and linear mode, by
accelerating the voltage to 20 kV. For sample preparation, 20 mg/mL
of 2-(4-hydroxy-phenylazo) benzoic acid (HABA) was used as a
matrix, and 1 mg/mL of sodium trifluoroacetate as the salt for
cationization were mixed with 6−7 mg/mL of the respective polymer
sample in 1,1,1,3,3,3-hexafluoro-2-isopropanol (HFIP) solution.16 The
melting points of the oligoamides were measured by differential
scanning calorimetry using a TA-Instruments Q1000 DSC. The
heating rate was 10 °C/min. Wide-angle X-ray diffraction (WAXD)
was performed using a Bruker D8 Advance and Cu Kα radiation, with
a wavelength of λ = 0.154 nm.15 Electron spray ionization mass
spectrometry (ESI-MS) was performed on a Thermo Scientific LTQ
Orbitrap mass spectrometer in positive ion mode. For sample
preparation approximately 1 mg/mL was dissolved in HFIP.
for toluene, diethyl sebacate, dimethyl terephthalate, and diamines, all
chemicals were used without further purification. Toluene was dried by
solvent purification system (SPS). Diethyl sebacate (DES) was dried
using calcium hydride and vacuum distilled. Dimethyl terephthalate
(DMTP) was recrystallized from methanol and dried in a vacuum
oven at T = 40 °C. 1,8-Diaminooctane (DAO) was purified by
sublimation. p-Xylylenediamine (p-XD) was purified by short-path
distillation using a Kugelrohr. Immobilized cutinase on Lewatit
(icutinase with enzyme loading 130 mg/g) and CLEA cutinase were
prepared as reported previously.7
Procedures of Enzymatic Polymerization of Aliphatic and
Aromatic Monomers and Enzymatic Reaction of Aromatic
Diamine and Diester. Enzymatic reactions were carried out via two
different reaction conditions: one-step and two-step reactions.
One-Step Enzymatic Synthesis. A total of 5 mL of dried toluene
was added to a mixture of 0.1 g of immobilized enzyme (CAL-B,
icutinase, or CLEA cutinase), diester (2.5 mmol), diamine (2.5 mmol),
and 0.5 g of dried molecular sieves. The mixture was stirred at 90 °C,
100 rpm for 48 h, under a N2 atmosphere. After cooling to room
temperature, toluene was removed by rotary evaporation. An aliquot
RESULTS AND DISCUSSION
■
1
from the residue was analyzed by H NMR in TFA-d for conversion
The enzymatic synthesis of aliphatic−aromatic oligoamides was
performed by polycondensation of DES and p-XD, or DMTP
and DAO in organic solvents using CAL-B, icutinase, or CLEA
cutinase as catalyst. Thermal stability and activity assays of
icutinase, CLEA cutinase,7 and CAL-B7,13,14 were previously
reported. Furthermore, we showed in our previous work that
CLEA cutinase possesses good catalytic activity toward aliphatic
diamines and diesters at 70 °C.7 However, no amide bonds
were formed at 70 °C, when the combination of aliphatic and
aromatic monomers was used, not even after 4 days. Therefore,
in this study, all reactions were performed at 90 °C for 2 days.
Optimization of the reaction was tried by longer reaction time
up to 4 days. However, the conversion of the reactions for 4
days remained the same as for reactions of 2 days.
determination. Formic acid (10 mL) was added to the rest of the
residue to dissolve the products. For the reactions catalyzed by CAL-B
or icutinase, the next step was filtration to separate the products from
the enzyme beads. For the reactions catalyzed by CLEA cutinase,
centrifugation was carried out to separate CLEA cutinase from the
product solution. All formic acid solutions containing products were
collected. Subsequently, for formic acid containing oligoamides,
precipitation in isopropanol and subsequent centrifugation were
carried out to isolate the oligoamide products. Precipitation of the
products of the reaction of DMTP and p-XD was performed in cold
CH3OH/H2O at 1:1 v/v ratio and centrifugation was carried out to
isolate the products. Furthermore, all synthesized products were dried
in a vacuum oven at 40 °C overnight.
Two-Step Enzymatic Synthesis. A total of 5 mL of diphenyl
ether was added to a mixture of 0.1 g of immobilized enzyme (CAL-B,
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Figure 1. Conversion of the one-step and two-step reactions for (A) oligo(p-xylylene sebacamide) and (B) oligo(octamethylene terephthalamide)
using CAL-B, icutinase, or CLEA cutinase.
Control reactions were done to determine whether reactions
occur without the presence of enzyme, as well. In the control
reactions (one-step and two-step), between DES and p-XD or
between DMTP and DAO, no amide bonds were formed.
Therefore, the appearance of amide bonds after the addition of
CAL-B, icutinase, or CLEA cutinase indicates that the reaction
is indeed an enzymatic polymerization.
Conversions of the one-step and the two-step reactions are
shown in Figure 1; the highest conversion was achieved in the
reaction catalyzed by CAL-B in comparison to icutinase or
CLEA cutinase. Reduced pressure (two-step reaction) did not
result in a higher conversion. In the synthesis of oligo(p-
xylylene sebacamide) and oligo(octamethylene terephthala-
mide), CAL-B showed better catalytic activity (higher
conversion) than CLEA cutinase or icutinase for both the
one-step and the two-step reactions.
In our previous study,7 CLEA cutinase showed higher activity
toward long chain aliphatic diamine (DAO) and diester (DES)
and nearly the same catalytic activity as CAL-B. In this study, a
very low conversion was observed in reactions using a
combination of aliphatic and aromatic diamines or diesters
and icutinase or CLEA cutinase as catalyst. Most probably
cutinase has difficulties to accept aromatic substrates due to
their bulkiness.
Figure 2. (1) ATR-FTIR spectra and (2) sample pictures of oligo(p-
xylylene sebacamide) catalyzed by (a) CAL-B, (b) icutinase, and (c)
CLEA cutinase. (1d) ATR-FTIR spectrum of CLEA cutinase.
Enzymatic Polymerization of p-XD and DES. Oligo(p-
xylylene sebacamide) was successfully enzymatically synthe-
sized by CAL-B, icutinase, or CLEA cutinase as catalyst, which
is confirmed by the ATR-FTIR . In the spectra (see Figure
2(1)), the formation of amide bonds is clearly proven by the
peaks at 1635 and 1539 cm−1 (amide I and II bands,
respectively) and the signals from the intermolecular hydrogen
bonding centered at 3290 cm−1. Furthermore, the carbonyl
group of the ester is still slightly visible at 1735 cm−1, which
proves that the formed oligoamide has ester end groups.
The different types of immobilized enzymes used in the
performed reactions resulted in a different coloration of the
products. Products synthesized by CLEA cutinase showed a
yellowish color when compared to the products synthesized
with CAL-B or icutinase, as shown in Figure 2(2c). In Figure
2(1c), the peaks that belong to C−O (alcohol) at 1022 cm−1
and the stretching vibrations of O−H or N−H at region 3400−
3300 cm−1 are clearly visible. These peaks were not found in
the two other samples catalyzed by CAL-B or icutinase. The
same peaks were observed in the ATR-FTIR spectrum of
CLEA cutinase, Figure 2(1d), which indicates some of the
CLEA cutinase is trapped between the oligo(p-xylylene
sebacamide) chains. CLEA cutinase is a fine powder with
yellowish color, while icutinase beads are colorless and have an
average particle size of 315−1000 μm. As for the separation and
purification of the products, formic acid is used as a solvent we
can observe that some of the yellowish color of CLEA cutinase
is mixed with the product. In the subsequent precipitation,
CLEA cutinase is trapped in the product and the peaks
belonging to CLEA cutinase are seen in the ATR-FTIR
spectrum; Figure 2(1c).
1
Figure 3 represents the H NMR spectra of the reaction
mixture of p-XD and DES after 2 days of reaction in both the
one-step and two-step reactions catalyzed by CAL-B; the acid
end group peak is clearly observed at 2.48 ppm. This indicates
that hydrolysis occurred, regardless of the drying conditions
used. Hydrolysis most probably occurs due to the residual
water content of CAL-B. After isolation of the product from the
reaction mixture, the peak at 2.48 ppm was no longer
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to the usage of formic acid for purification − it reacts with the
amine groups of the amine−amine end groups.24
From MALDI-ToF MS spectra, the end group of the
oligo(p-xylylene sebacamide) can be determined as correspond-
ing to M = x + (n × mass of repeating unit) + Na+,16 where n is
the degree of polymerization and x is the mass of the end
groups. In Table 1, the five different end groups, amine−amine,
amine−ester, amine−acid, ester−ester, and amide−amine are
summarized. The mass of the repeating unit oligo(p-xylylene
sebacamide) is 302.2 Da, x = 136.1 Da for amine−amine, x =
46 Da for amine−ester, x = 18 Da for amine−acid, x = 258.2
for ester−ester, and x = 164.1 for amide−amine . For example,
the mass for n = 2, amine−amine end group is 763.5 Da, found
at 763.32 Da in the MALDI-ToF spectrum (see Figure 4).
The maximal degree of polymerization (DPmax) was
determined by MALDI-ToF MS. The highest DPmax was
observed in oligo(p-xylylene sebacamide), which was catalyzed
by CLEA cutinase, in both one-step and two-step reactions. For
this reason, CLEA cutinase could be a good candidate for
enzymatic catalysis for aliphatic or aromatic monomers if the
conversion can be increased (as compared to CAL-B).
The most dominant end group in all products was the
amine−amine end group. Sodium was the most abundant
adduct that was observed in the MALDI-ToF spectra.
Additionally, we noticed differences between products synthe-
sized in the one-step and the two-step reactions (see
Supporting Information, Figures S2−S6). The product
synthesized in the one-step reaction had no ester−ester end
group and a slightly lower DPmax. In the one-step reaction the
ethanol produced by the reaction remains mainly in the
reaction mixture and can cause a transesferification reaction
between the ester groups (ester−ester or amine−ester) and
1
Figure 3. H NMR spectra of the reaction mixture of p-XD and DES
after a 2 d catalyzed by CAL-B in the (a) one-step and (b) two-step
reactions.
observable. For the reactions catalyzed by icutinase or CLEA
cutinase, no peak at 2.48 ppm was observed due to very low
conversion.
Analysis of the end groups of oligo(p-xylylene sebacamide)
1
1
can be performed by H NMR. From the H NMR spectra of
oligo(p-xylylene sebacamide) (Figure S1, Supporting Informa-
tion), it becomes obvious that at least two end groups are
present; amine−amine and amine−ester end groups. Further
analysis with MALDI-ToF MS revealed that oligo(p-xylylene
sebacamide) has five different end groups, which are amine−
amine, amine−ester, amine−acid, ester−ester, and amide−
amine, see Table 1. The amide−amine end group is formed due
Table 1. Different Microstructures and End Groups of Oligo(p-xylylene sebacamide)
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Figure 4. MALDI-ToF MS spectrum of oligo(p-xylylene sebacamide)
synthesized in the two-step reaction by CLEA cutinase.
Figure 5. XRD spectra of oligo(p-xylylene sebacamide) synthesized
using (a) CAL-B, (b) icutinase, and (c) CLEA cutinase.
ethanol. Not only ethanol gives rise to side reactions, but also
the presence of water can cause hydrolysis. Therefore, the
presence of ethanol and water in the reaction can cause
competition between transesterification, hydrolysis, and poly-
merization in the one-pot reaction. Due to this reason the
enzyme is less efficient and the enzymatic polymerization
resulted in a lower DPmax. In the two-step reactions ethanol and
water are removed by vacuum to a high extent. Therefore, a
higher DPmax was achieved in the two-step reaction, as shown in
Table 2.
Supporting Information. Furthermore, we observed a weight
loss of the samples at around 10−15% after DSC measure-
ments. The weight loss probably occurs due to some water,
diphenyl ether, or other small molecules trapped in the samples
that evaporate during heating up to 258 °C.
Enzymatic Polymerization of DMTP and DAO. Oligo-
(octamethylene terephthalamide) was successfully synthesized
by the polymerization of DMTP and DAO catalyzed by CAL-B
or CLEA cutinase in a one-step and a two-step reaction,
respectively. In reactions catalyzed by icutinase, a low
conversion of the amide bond was observed only for the one-
step reaction (∼3%). In general, the polymerization of DMTP
and DAO resulted in lower conversions than the polymer-
ization of p-XD and DES. This could be due to the conjugated
system of carboxylate group and benzene ring in DMTP, which
can hinder the formation of the acyl-enzyme intermediate from
DMTP.
Table 2. DPmax of Oligo(p-xylylene sebacamide) and
Oligo(octamethylene terephthalamide)
one-step reaction; two-step reaction;
oligomer
catalyst
CAL-B
DPmax (MALDI)
DPmax (MALDI)
oligo(p-xylylene
sebacamide)
10
14
icutinase
8
8
CLEA
cutinase
15
15
1H NMR analysis of oligo(octamethylene terephthalamide)
indicated the existence of at least one end group; amine−ester
(Supporting Information, Figure S9). Furthermore, end group
analysis assessed by MALDI-ToF MS showed that oligo-
(octamethylene terephthalamide) has three different end
groups: amine−ester, amine−amine, and ester−ester, see
Table 3. The amine−amide end group was not observed,
although we used formic acid to dissolve and isolate the
oligoamide. This is probably due to a more difficult reaction
between formic acid and aliphatic amine than the reaction with
an aromatic amine. It was already shown in literature that the
reaction between formic acid and aliphatic amine proceeds at
temperatures as high as 150, 250, or 350 °C.25
The most dominant end group found in both the one-step
and the two-step reaction was the ester−ester end group and
the highest DPmax achieved with this end group was 6. The end
group determination was performed as explained previously in
the synthesis of oligo(p-xylylene sebacamide), corresponding to
the equation: M = x + (n × mass of repeating unit) + Na+,16
where x is the mass of the end groups (amine−ester, amine−
amine, and ester−ester), as shown in Table 3. The mass of the
repeating unit oligo(octamethylene terephthalamide) is 274.1
Da, x = 32 Da for amine−ester, x = 144.2 Da for amine−amine,
and x = 194.1 Da for ester−ester end group. As an example for
n = 1, the mass of the ester−ester end group is 491.2 Da and
oligo(octamethylene
terephthalamide)
CAL-B
6
a
6
6
a
6
icutinase
CLEA
cutinase
a
Results could not be defined.
XRD measurement of the oligo(p-xylylene sebacamide)
synthesized in a two-step reaction was performed to evaluate
the crystallinity of the products. Similar XRD spectra pattern
were observed for the three different samples catalyzed by
CAL-B, icutinase, or CLEA cutinase, respectively. From the
XRD spectra shown in Figure 5, we can conclude that oligo(p-
xylylene sebacamide) is a semicrystalline oligomer.
DSC measurements were performed in order to determine
the melting temperature of oligo(p-xylylene sebacamide). The
melting temperature varied between 223−230 °C (Figure S7,
Supporting Information). The highest melting temperature was
observed for oligo(p-xylylene sebacamide) catalyzed by CLEA
cutinase, which is in agreement with the MALDI-ToF MS
results, for oligo(p-xylylene sebacamide) catalyzed by CLEA
cutinase, which has the highest DPmax. A glass transition
temperature (Tg) could be observed at 59 °C in the second
heating curve of a DSC scan, as shown in Figure S8 in the
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Table 3. Different Microstructures and End Groups of Oligo(octamethylene terephthalamide)
found at 491.15 Da in the MALDI spectrum, see Figure S10 in
Supporting Information.
Scheme 2. Enzymatic Reaction between Aromatic Diamine
and Diester
XRD measurements were performed for two samples
synthesized in the one-step reaction catalyzed by CAL-B and
by CLEA cutinase (Figure S11 in Supporting Information).
Oligo(octamethylene terephthalamide) catalyzed by CAL-B
resulted in a product with a higher crystallinity than
oligo(octamethylene terephthalamide) catalyzed by CLEA
cutinase. This result was in a good agreement with the DSC
analysis; two melting temperatures at 184 and 216 °C were
observed for the sample catalyzed by CAL-B (Figure S12 in
Supporting Information) and no melting temperature was
observed for the sample catalyzed by CLEA cutinase.
Crystallinity and melting temperatures were not observed in
the oligo(octamethylene terephthalamide) catalyzed by CLEA
cutinase due to the very low conversion of oligo(octamethylene
terephthalamide) and the contamination with CLEA cutinase
residues.
Enzymatic Reaction of DMTP and p-XD. In the reaction
between DMTP and p-XD, CAL-B, icutinase, or CLEA cutinase
catalyzed the formation of amide bonds but no oligomers could
be observed. The reaction between DMTP and p-XD was
carried out in both one-step and two-step reactions. The
highest conversion (∼19%) was observed in the one-step
reaction catalyzed by CAL-B. Reduction of the pressure in the
two-step reaction did not result in longer chains of amide
product.
The enzymatic reaction of DMTP and p-XD resulted in
products with ester−ester end groups or product 1 (see scheme
2), as detected by ESI-MS and MALDI-ToF MS (Figures S14
and S15 in Supporting Information). Small amounts of ester−
acid end group (product 2) were found in the product
catalyzed by CAL-B, as shown in Figure 6. Unfortunately, CAL-
B and cutinase did not catalyze the formation of oligoamide
from the reaction of DMTP and p-XD. The oligomer growth
was stopped at a certain length probably due to steric hindrance
of the product that already formed in the pocket of the active
site of the enzyme.
The use of CLEA cutinase in the reaction of p-XD and
DMTP did not contaminate the product, unlike in the
syntheses of oligo(p-xylylene sebacamide) and oligo-
(octamethylene terephthalamide). Figure S16 (Supporting
Information) shows the ATR-FTIR spectra without CLEA
cutinase contamination. This could occur because products 1
and 2 have very short chains; thus, no CLEA cutinase got
trapped in the chains. Therefore, it can be concluded that for
the synthesis of oligoamides or polyamides the use of
immobilized enzyme on beads is more suitable than in the
form of CLEA.
CONCLUSION
■
The enzymatic synthesis of oligo(p-xylylene sebacamide) and
oligo(octamethylene terephthalamide) was successfully per-
formed with CAL-B, icutinase, or CLEA cutinase. Higher
conversion is achieved using CAL-B as catalyst. Although
reactions using CLEA cutinase as catalyst showed lower
conversion than reactions using CAL-B they resulted in the
same or even higher DPmax, which indicates the clear potential
of CLEA cutinase for enzymatic polymerizations. The reactions
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dx.doi.org/10.1021/bm400243a | Biomacromolecules 2013, 14, 1600−1606

Biomacromolecules
Article
ACKNOWLEDGMENTS
■
The authors wish to thank the Ubbo Emmius Programme of
the University of Groningen for financial support, Novozymes
(Denmark) for supplying Fusarium solani pisi cutinase, Lanxess
(Belgium) for supplying Lewatit VP OV 1600, T. D.
Tiemersma-Wegman for ESI-MS measurement, and Jakob
Baas for WAXD measurements.
REFERENCES
■
557.
(1) Levchik, S. V.; Weil, E. D.; Lewin, M. Polym. Int. 1999, 48, 532−
(2) Zulfiqar, S.; Ishaq, M.; Ilyas Sarwar, M. Adv. Polym. Technol. 2010,
29, 300−308.
(3) Trigo-Lop
́
ez, M.; Estev
́
ez, P.; San-Jose,
́
N.; Gom
́
ez-Valdemoro,
A.; García, F. C.; Serna, F.; de la Pena, J. L.; García, J. M. Recent Pat.
̃
Mater. Sci. 2009, 2, 190−208.
(4) Yang, S. H.; Fu, P.; Liu, M. Y.; Wang, Y. D.; Li, Z. P.; Zhao, Q. X.
Express Polym. Lett. 2010, 4, 346−354.
1
Figure 6. H NMR spectra of the product of the reaction between p-
XD and DMTP in the two-step reaction catalyzed by (a) CAL-B and
(b) CLEA cutinase.
(5) Uyama, H.; Kobayashi, S., Enzymatic Synthesis of Polyesters via
Polycondensation. In Enzyme-Catalyzed Synthesis of Polymers;
Kobayashi, S., Ritter, H., Kaplan, D., Eds.; Springer: Berlin, Heidelberg,
2006; Vol. 194, pp 133−158.
(6) Cheng, H. N., Enzyme-Catalyzed Synthesis of Polyamides and
Polypeptides. Biocatalysis in Polymer Chemistry; Wiley-VCH Verlag
GmbH & Co. KGaA: New York, 2010; pp 131−141.
(7) Stavila, E.; Arsyi, R. Z.; Petrovic, D. M.; Loos, K. Eur. Polym. J.
2013, 49, 834−842.
(8) Schwab, L. W.; Kloosterman, W. M. J.; Konieczny, J.; Loos, K.
Polymers 2012, 4, 710−740.
(9) Schwab, L. W.; Kloosterman, W. M. J.; Konieczny, J.; Loos, K. J.
Renewable Mater. 2013, 1, 73−78.
of aromatic diamine and diester (p-XD and DMTP), using
CAL-B or CLEA cutinase as catalysts resulted in short chain
amide product.
The highest DPmax of 15 for oligo(p-xylylene sebacamide)
with a melting temperature of 230 °C was observed in the
enzymatic polymerization of p-XD and DES in the two-step
reaction using CLEA cutinase as a catalyst. The higher DPmax
was observed in the two-step reaction process due to the better
removal of water and ethanol by pressure reduction. By
removing the water, hydrolysis during polymerization is
prevented, resulting in an increased amount of ester−ester
end groups. Oligo(p-xylylene sebacamide) synthesized in a two-
step reaction process had five different end groups: amine−
amine, amine−ester, amine−acid, ester−ester, and amide−
amine.
The highest DPmax of 6 for oligo(octamethylene tereph-
thalamide) with two melting temperatures at 186 and 218 °C
was observed in the enzymatic polymerization of DMTP and
DAO in the two-step reaction using CAL-B as a catalyst. The
oligo(octamethylene terephthalamide) synthesized in one-step
or two-step reactions had three different end groups: amine−
amine, amine−ester, and ester−ester. By using an aromatic
diester (DMTP) the enzymatic polymerization is hindered due
to the conjugated system between the benzene ring and the
ester group. In the one-step, as well as the two-step reaction,
ester−ester end groups can be observed. Therefore, it can be
concluded that the presence of water in the reaction cannot
cause hydrolysis during polymerization.
(10) Gotor, V.; Brieva, R.; Gonzal
1991, 47, 9207−9214.
́
ez, C.; Rebolledo, F. Tetrahedron
(11) Sergeeva, M.; Mozhaev, V.; Rich, J.; Khmelnitsky, Y. Biotechnol.
Lett. 2000, 22, 1419−1422.
(12) Gotor, V. Enzymatic Aminolysis and Ammonolysis Reactions. In
Enzymes in Action; Zwanenburg, B., Mikołajczyk, M., Kiełbasin
Eds.; Springer: Netherlands, 2000; Vol. 33, pp 117−132.
́
ski, P.,
(13) Stavila, E.; Loos, K. Tetrahedron Lett. 2013, 54, 370−372.
(14) Schwab, L. W.; Kroon, R.; Schouten, A. J.; Loos, K. Macromol.
Rapid Commun. 2008, 29, 794−797.
(15) Poojari, Y.; Clarson, S. J. Macromolecules 2010, 43, 4616−4622.
(16) Ragupathy, L.; Ziener, U.; Dyllick-Brenzinger, R.; von Vacano,
B.; Landfester, K. J. Mol. Catal. B: Enzym. 2012, 76, 94−105.
(17) Baum, I.; Elsasser, B.; Schwab, L. W.; Loos, K.; Fels, G. ACS
̈
Catal. 2011, 1, 323−336.
(18) Sharma, B.; Azim, A.; Azim, H.; Gross, R. A.; Zini, E.; Focarete,
M. L.; Scandola, M. Macromolecules 2007, 40, 7919−7927.
(19) Palsule, A. S.; Poojari, Y. Polymer 2010, 51, 6161−6167.
(20) Poulhes
Tetrahedron: Asymmetry 2012, 23, 867−875.
(21) Poulhes, F.; Mouysset, D.; Gil, G.; Bertrand, M. P.; Gastaldi, S.
̀
, F.; Mouysset, D.; Gil, G.; Bertrand, M. P.; Gastaldi, S.
̀
Polymer 2012, 53, 1172−1179.
ASSOCIATED CONTENT
* Supporting Information
■
(22) Uyama, H.; Yaguchi, S.; Kobayashi, S. Polym. J. 1999, 31, 380−
S
383.
1H NMR spectra, MALDI-ToF MS spectra, DSC curve, and
XRD spectra of oligo(p-xylylene sebacamide) or oligo-
(octamethylene terephthalamide); MALDI-ToF MS spectra,
ESI-MS chromatograms, and ATR-FTIR spectra of amide
products. This material is available free of charge via the
Internet at http://pubs.acs.org.
(23) Wu, X. Y.; Linko, Y. Y.; Seppala, J.; Leisola, M.; Linko, P. J. Ind.
̈
̈
Microbiol. Biotechnol. 1998, 20, 328.
(24) Jung, S. H.; Ahn, J. H.; Park, S. k.; Choi, J.-k. Bull. Korean Chem.
Soc. 2002, 23, 149−150.
(25) Katritzky, A. R.; Parris, R. L.; Ignatchenko, E. S.; Allin, S. M.;
Siskin, M. J. Prakt. Chem. 1997, 339, 59−65.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +31-503636867. E-mail: k.u.loos@rug.nl.
■
Notes
The authors declare no competing financial interest.
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