Biocatalytic synthesis of silicone polyesters

Article abstract of DOI:10.1021/bm100295z

The immobilized lipase B from Candida antarctica (CALB) was used to synthesize silicone polyesters. CALB routinely generated between 74-95% polytransesterification depending on the monomers that were used. Low molecular weight diols resulted in the highes

Full text of DOI:10.1021/bm100295z

1
818
Biomacromolecules 2010, 11, 1818–1825
Biocatalytic Synthesis of Silicone Polyesters
Mark B. Frampton, Izabela Subczynska, and Paul M. Zelisko*
Department of Chemistry and Centre for Biotechnology, Brock University, 500 Glenridge Avenue,
St. Catharines, Ontario, Canada
Received March 18, 2010; Revised Manuscript Received May 14, 2010
The immobilized lipase B from Candida antarctica (CALB) was used to synthesize silicone polyesters. CALB
routinely generated between 74-95% polytransesterification depending on the monomers that were used. Low
molecular weight diols resulted in the highest rates of esterification. Rate constants were determined for the
CALB catalyzed polytransesterifications at various reaction temperatures. The temperature dependence of the
CALB-mediated polytransesterifications was examined. A lipase from C. rugosa was only successful in performing
esterifications using carboxy-modified silicones that possessed alkyl chains greater than three methylene units
between the carbonyl and the dimethylsiloxy groups. The proteases R-chymotrypsin and papain were not suitable
enzymes for catalyzing any polytransesterification reactions.
Introduction
Polyesters are used commercially as fibers, plastics, and
chiral alcohols, although more recently they have gained
6
-8
popularity for the synthesis of polycarbonate materials,
9
-12
inorganic-organic hybrid silicone polymeric materials,
the
coatings and are typically synthesized by esterification or
transesterification reactions between acids/esters and alcohols,
as well as the reaction of alcohols with acid chlorides and
1
3
polymerization of polyhydroxyalkanoate block copolymers,
and for ring-opening polymerizations. Many of these enzyme-
mediated poly(trans)esterification reactions were tolerant to the
5,14
1
anhydrides. Silicones are desired for their industrial applications
1
5-17
presence of pendant functional groups in the monomers.
as well as their physicochemical properties (surface activity,
thermostability, chemical stability, low glass transition temper-
ature, etc.) and the properties that they can impart to organic
Typically, acids and vinyl or alkyl esters have been employed
as acyl donors, although O-carboxylic anhydrides have also been
5
2
,3
utilized.
polymers when included as comonomers. The basis for these
valuable properties lies in the strength and flexibility of the
Si-O bond, the ionic character of the siloxane bond, and the
Lipase-mediated copolymerizations of hybrid inorganic-
organic monomers have been receiving an increasing amount
of attention. The immobilized lipase B from Candida antarctica,
sold under the name Novozym 435, has been the enzyme of
3
low interactive forces between the geminal dimethyl groups.
The increased length of the Si-O (1.64 Å) and Si-C (1.87 Å)
bonds compared to C-O (1.41 Å) and C-C (1.53 Å) bonds
permits a localized reduction in the steric bulk that results in a
9
,12,18
choice for synthesizing silicone containing polyesters,
1
0
11
polyamides, and polyimides. Silicone diols have been
transesterified with a series of diacids of increasing molecular
weight. Polymer molecular weight could be improved upon
by refluxing in hexanes compared to the application of vacuum
for bulk preparations. The synthesis of organosiloxane copoly-
mers from a variety of aliphatic diols illustrated the dependence
3
higher degree of rotational freedom. The incorporation of
1
2
organic groups within the backbone of the silicone chain can
change the physical characteristics of the silicone polymer.
Silicone materials are typically synthesized using transition
metal catalysts such as tin, titanium, or platinum, although
peroxide-induced free radical reactions and condensation reac-
tions employing acetoxy or alkoxy groups are also useful
n w
of the M and M of the final polymer on the molecular weight
9
of the diol monomer. Applied vacuum, enzyme loading, and
the specific activity of the lipase affected the molecular weights
of the resulting copolymers. While applied vacuum had little
4
methods. However, if cross-linked silicone materials are to be
used in biomedical or agrichemical applications, the use of
potentially toxic metals can be limiting. One potential route to
the design and synthesis of biocompatible materials is through
enzymatic or biomimetic catalysis.
effect on M
for higher M
reaction. Both enzyme loading and the specific activity of
the lipase were also found to increase the M of the polymers.
w
during the initial stages of the reaction, it did allow
w
to be achieved during the later stages of the
w
Biocatalysis can offer a mild route to synthetic intermediates
that may otherwise be unobtainable. Because enzymes are
themselves chiral species, they often show a preference for a
specific enantiomer, although this is not always the case.
Novozym 435, while showing a preference to L-lactide O-
carboxylic anhydride, was still capable of polymerizing the
D-isomer albeit with a 2.5-fold reduction in the rate of
The copolymerization of an amine functionalized silicone with
8
diethyl adipate and 1,8-octanediol has recently been described.
The distribution of units along the polymer chain was deter-
1
mined to be random by H NMR spectroscopy. The synthesis
of siloximide copolymers required the active site of an im-
mobilized lipase to mediate ring closure to form the diphthalate
bridges between repeating aminopropyl-functionalized silicone
5
processing.
11
repeating units. Silanol-terminated polydimethylsiloxane (PDMS)
was quantitatively esterified with vinyl methacrylate in 2 h under
solvent-free conditions, giving rise to methacrylate-terminated
Lipases are prevalent throughout nature and have been
isolated from a variety of bacteria, plant, fungal, and mammalian
sources. Lipases have typically been used for the resolution of
1
9
PDMS.
There is a growing trend toward the design and development
of environmentally benign synthetic methods, as well as the
*
To whom correspondence should be addressed. Tel.: 905-688-5550
x.4389. Fax: 905-682-9020. E-mail: pzelisko@brocku.ca.
10.1021/bm100295z
 2010 American Chemical Society
Published on Web 06/17/2010

Biocatalytic Synthesis of Silicone Polyesters
Biomacromolecules, Vol. 11, No. 7, 2010 1819
ethylsiloxane (MW 1000 g/mol), 1,3-carboxypropyl-1,1,3,3-tetrameth-
yldisiloxane (CPr-TMDS), and 1,1,3,3-tetramethyldisiloxane were
obtained from Gelest, Inc. (Morristown, Pennsylvania). p-Toluene
sulfonic acid (pTsOH) was obtained from Eastman Kodak Company
(
Rochester, NY). Isooctane and pentane were obtained from Caledon
Chemicals (Georgetown, Ontario, Canada). Chloroform-d (99.8%
deuterated) was a product of Cambridge Isotope Laboratories, Inc.
(
Landover, Maryland). Toluene was obtained from EMD (Gibbstown,
NJ). Diethyl ether was acquired from Anachemia (Montreal, Quebec,
Canada). Distilled water was used for all preparations. All reagents
were used as received without further modification or purification.
Nuclear Magnetic Resonance Spectroscopy (NMR). Solution phase
NMR spectra were acquired using a Bruker Avance AV-300 (300 MHz
1
13
29
for H; 77.5 MHz for C; 59.6 MHz for Si) spectrometer. Spectra
Figure 1. Generalized reaction scheme for the enzyme-mediated
synthesis of silicone polyesters.
1
13
3
were referenced to CDCl (7.26 ppm for H and 77.0 ppm for C) as
an internal standard; Si NMR spectra were referenced to TMS (0.0
ppm).
29
Fourier-Transform Infrared Spectroscopy (FTIR). FTIR spectros-
copy was performed on a Mattson Research Series infrared spectrometer
in transmittance mode on samples prepared as thin films on KBr. Each
FTIR spectrum was acquired as 32-64 scans at 2 cm resolution.
Spectra were analyzed using the Winfirst software platform.
MALDI-TOF Mass Spectrometry. Molecular weight determination
incorporation of materials derived from renewable resources into
current chemical processes. Biotransformations of silicon-based
materials have eliminated the need for transition metals, harsh
acids and bases for catalysis, and extremes of temperature and
pressure. Biotransformations have typically been performed at
lower temperatures (50-90 °C) and at atmospheric or reduced
pressures (particularly at the later stages of the reaction to
facilitate oligomerization of monomers and dimers) when
-1
n w
of the end products of enzyme-mediated polymerizations, M and M ,
were determined using MALDI-TOF mass spectrometry. MALDI-TOF
MS spectra were acquired using a Bruker Autoflex spectrometer
operated in the positive reflectance mode. Samples were prepared as
solutions in THF containing dithranol as the matrix.
8,9,18
comparedtotraditionalroutesforperformingsimilarreactions.
The elimination of solvents from these reactions is also
becoming increasingly popular, although in some instances
solvents cannot be avoided as a result of solubility consider-
Synthesis of 1,3-Bis(3-hydroxypropyl)-1,1,3,3-tetramethyldisilox-
ane (3HP-TMDS, 1). It is known that the hydrosilylation of Si-H
functional groups with allyl alcohol can lead to potential Si-O
couplings. This was minimized using a modification of the method
1
8
ations.
R-Chymotrypsin is a serine protease that is catalytically
homologous to CALB and has been shown to be capable of
2
6
reported by Zhang and Laine. A round bottomed flask was charged
with allyl alcohol (3.86 mL, 56.8 mmol), Karstedt’s catalyst (20 µL),
and 20 mL of toluene and stirred at ambient temperature for 5 min
after which time 1,1,3,3-tetramethyldisiloxane (5.0 mL, 28.3 mmol)
was added and the reaction was refluxed for 4 h. Product formation
was monitored by FTIR operated in the transmittance mode by
monitoring the disappearance of the Si-H resonance typically located
2
0,21
mediating ester synthesis,
as well as chemical transforma-
2
2,23
tions not associated with peptide hydrolysis.
Papain on the
other hand is neither structurally, nor catalytically homologous
to the serine hydrolases, but instead contains a catalytic cysteine-
histidine dyad that is used for peptide hydrolysis. Papain has
been used to mediate the polymerization of L-tyrosine ethyl ester
hydrochloride, diethyl glutamate, and leucine ethyl ester under
buffered conditions to yield poly(tyrosine), poly(glutamic acid),
-1
at ∼2110-2160 cm . Once this peak was no longer visible, the
reaction was allowed to reflux for an additional hour to ensure the
complete consumption of starting materials. The reaction was decol-
orized by adding activated carbon and an excess of pentane while
stirring for 3 h. The crude reaction mixture was filtered through Celite
545, washed with pentane, and the solvent removed in vacuo to yield
2
1,24,25
and poly(leucine).
In the current work we describe results relating to the
screening of immobilized and free lipases, as well as R-chy-
motrypsin and papain, for the synthesis of silicone polyesters
(
Figure 1). In some systems the need to use solvents was
a clear colorless liquid in 73-93% yield. The product was confirmed
1
completely eliminated, but for others this was not possible.
Kinetic and thermodynamic parameters for the lipase-mediated
polytransesterifications were determined employing H NMR
spectroscopy. Silicone polyesters were characterized by NMR,
Fourier transform infrared spectroscopy (FT-IR), and matrix-
assisted laser desorption ionization time-of-flight (MALDI-TOF)
mass spectrometry.
by H NMR (CDCl
3
): 0.09 ppm (s, 12H, SiMe
2
), 0.53 ppm (t, 4H,
-CH -CH -Si), and
2
-Si); C NMR ( H decoupled,
HO-CH
.60 ppm (t, 4H, HO-CH
CDCl ): 0.0 ppm (SiMe ), 13.94 ppm (HO-CH
ppm (HO-CH -CH -CH -Si), 65.59 ppm (HO-CH
2
-CH
2
-CH
2
-Si), 1.60 ppm (m, 4H, HO-CH
2
2
1
2
1
3
1
3
2
-CH
2
-CH
3
2
2
-CH
2
-CH
2
-Si), 26.58
2
9
2
2
2
2
-CH
2
-CH
2
-Si); Si
1
-1
3
NMR ( H decoupled, CDCl ): 8.22 ppm. FTIR (neat film, KBr, 2 cm
resolution): 703, 801, 1032, 1087, 1260, 1412, 2933, 2961, 3329 cm-1.
Synthesis of r,ω-Bis(3-hydroxypropyl)-polydimethylsiloxane (3HP-
PDMS, 2). A round bottomed flask was charged with allyl alcohol (1.0
mL, 16 mmol), 20 µL of Karstedt’s catalyst, and 10 mL of toluene.
After stirring at ambient room temperature for 5 min, hydride-terminated
polydimethylsiloxane (5.0 mL, 8 mmol) was added and the reaction
mixture was refluxed. Product formation was monitored by FTIR
following the disappearance of the Si-H resonance at ∼2110-2160
Experimental Section
Materials. Lipase from Candida antarctica (CALB) (EC.3.1.1.3,
1
(
0000 U/g), lipase Type VII from Candida rugosa (1180 U/mg solid)
LCR), lipase type I from wheat germ (EC.3.1.1.3, 7.9 U/mg solid, 8.2
U/mg protein), R-chymotrypsin type II from bovine pancreas (EC.3.4.2.1,
3.9 U/mg solid), hydride-terminated polydimethylsiloxane (M 580
g/mol), platinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane complex
Karstedt’s catalyst) in xylenes, and tetramethylsilane (TMS) were
acquired from Sigma Aldrich (Oakville, Ontario, Canada). Papain from
Carica papaya (EC. 3.4.22.2, 3.36 U/mg) was obtained from Fluka
Analytical (Oakville, Ontario, Canada). Allyl alcohol was obtained from
Alfa Aesar (Ward Hill, Massachusetts). R,ω-Carboxydecyl polydim-
-
1
8
n
cm . Once this peak was no longer visible, the reaction was allowed
to reflux for an additional hour. The reaction was decolorized by adding
activated carbon and an excess of pentane while stirring for 2 h. The
reaction mixture was filtered through Celite 545, washed with pentane,
(
and the solvent was removed in vacuo to yield a clear colorless liquid
1
in 63-94% yield. The product was confirmed by H NMR (CDCl
3
):
2 2 2 2
0.05 ppm (s, 56H, SiMe ), 0.54 ppm (t, 4H, HO-CH -CH -CH
-Si),

1
820 Biomacromolecules, Vol. 11, No. 7, 2010
Frampton et al.
1
CH
.60 ppm (m, 4H, HO-CH
2
-CH
2
-CH
2
-Si), and 3.60 ppm (t, 4H, HO-
-Si); C NMR ( H decoupled, CDCl ): 0.12, 1.08, 1.18
-Si), 26.58 ppm (HO-CH
Table 1. Lipases and Proteases Used in this Work, the Specific
Activity as Stated by the Manufacturer, and Reaction
Temperatures Used during this Study
1
3
1
2
-CH
2
-CH
), 13.94 ppm (HO-CH
-Si), 65.59 ppm (HO-CH
2
3
ppm (SiMe
CH -CH
decoupled, CDCl
2
2
-CH
2
-CH
2
2
-
2
9
1
2
2
2
-CH
2
2
-CH -Si); Si NMR ( H
enzyme
specific activity
temp (°C)
3
): 7.86 ppm, -21.19 ppm, and -21.93 ppm. FTIR
lipase (C. antarctica)
lipase (C. rugosa)
lipase (wheat germ)
R-chymotrypsin (B. taurus)
papain (C. papaya)
12000 U/g
1180 U/mg
7.9 U/mg
83.9 U/mg
3.36 U/mg
35, 50, and 70
-
1
(
neat film, KBr, 2 cm resolution): 3329, 2961, 1412, 1260, 1087,
35
35
35
35
-
1
1
032, 801, 703 cm .
Synthesis of 1,3-Bis(carboxypropyl)-1,1,3,3-tetramethyldisiloxane
dimethyl ester (CPr-TMDS-DME, 4). A 250 mL oven-dried round
bottomed flask was charged with CPr-TMDS (4.22 g, 13.8 mmol), 20
mL of methanol, and 106 µmol (7 mol %) p-toluene sulfonic acid, and
then refluxed for 4 h. After cooling to room temperature, the methanol
was removed in vacuo. The remaining residue was extracted twice with
Table 2. Degree of Esterification (Monomer Conversion) for
Enzyme-Free Control Reactions is Dependent on the Acyl Donor
Employed and the Reaction Temperature
1
0 mL of Et
with 5 mL of saturated carbonate solution, and 5 mL of brine. The
ethereal layer was dried over MgSO and the solvent removed in vacuo
2
O, followed by two washings with 10 mL of water, once
monomer conversion using
acyl donor temperature (°C) 3HP-TMDS (%) 3HP-PDMS (%)
4
3
35
9
41
0
3
27
0
to give a clear and colorless liquid in 77% recovery. It should be noted
that at lower pTsOH concentrations (0.5-2%) that the corresponding
70
4
70
silanol was often present as a byproduct in low concentrations (∼10%).
1
Spectroscopic analysis: H NMR (CDCl
3
): 0.0 ppm (s, 12H, SiMe
2
,
methodologies. Biocatalysis offers access to products or inter-
mediates without the use of harsh reaction conditions such as
extremes of temperature, pressure, or potentially toxic catalysts.
This report describes the synthesis and characterization of AB
block silicone polyesters produced using enzymatic catalysis.
Although certain silicones, in particular, cyclic species, have
been shown to be detrimental to enzymes, higher molecular
weight silicone species have been shown to protect enzymes
from denaturation and allowed them to maintain their biological
disiloxane), 0.53 ppm (m, 4H, CH
ppm (m, 4H, CH -CH -CH -SiMe
SiMe ), 3.66 ppm (s, 6H, OMe); C NMR ( H decoupled, CDCl
.23 ppm (SiMe
CH -CH -CH -SiMe
2
-CH
2
-CH
2
-SiMe
2
, disiloxane), 1.64
2
2
2
2
1
), 2.32 ppm (t, 4H, CH
2
-CH -CH -
2
2
3
1
2
3
):
), 19.310 ppm
0
(
2
), 18.01 ppm (CH
2
-CH
2
-CH
2
-SiMe
2
2
2
2
2
), 37.46 ppm (CH
2
-CH
2
-CH
2
-SiMe
2
), 51.36 ppm
):
); FTIR (neat film, KBr, 2 cm resolution):
532, 2954, 2900, 2879, 1741, 1437, 1254, 1205, 1058, 1020, 885,
2
9
1
(
(CdO)OMe), 174.06 ppm (CdO); Si NMR ( H decoupled, CDCl
3
.32 ppm (Me Si-O-SiMe
2 2
-
1
7
3
8
-
1
39, 796 cm
.
2
9-32
Silicone Polyester Synthesis. Silicone polyesters were prepared both
activity.
It has even been demonstrated that lipases
as neat mixtures and in solution/suspension as necessary using either
acrylic resin immobilized lipase from C. antarctica or free lipases or
proteases. Neat preparations: Carboxy-functionalized silicones were
combined directly with hydroxy-functionalized silicones in a 1:1 ratio
entrapped within silicone elastomers were able to perform
33,34
esterification reactions.
These entrapped lipases were found
to be more active in silicones than in purely hydrocarbon
systems. Given that lipases tend to be more active in hydro-
phobic environments, it is perhaps not surprising that they are
quite active in silicone systems that are in and of themselves
(
approximately 500 µL reaction volume) in a 5 mL round bottomed
flask and preheated to 35 or 70 °C. Immobilized enzymes were added
to the reaction mixture and stirred for 72 h while stirring at 150 rpm
with the application of vacuum after each 24 h period for approximately
35
quite hydrophobic.
Silicone polyesters were synthesized by enzymatic means and
characterized by NMR, FTIR, and MS techniques. Immobilized
and free enzymes were screened as potential candidates for this
process. Candida antarctica lipase B was immobilized on acrylic
resin, while the free enzymes were prepared from lyophilized
powders. Enzyme-free control reactions were performed using
a 1:1 ratio of monomers to determine the background rate of
esterification between 1 or 2 and 1,3-bis(3-carboxypropyl)-
1,1,3,3-tetramethyldisiloxane (CPr-TMDS, 3; Table 2). In the
absence of any enzyme catalyst, the background rate of
monomer conversion ranged from 25-40% at 70 °C when 3
was used as the acyl donor, suggesting that AB dimers and ABA
(BAB) trimers were being formed. The same acyl donor was
employed by Poojari et al., but it was unclear if a similar
phenomenon was observed when alkane diols were employed
1
-2 min. The reactions were terminated with the addition of 2-3 mL
of diethyl ether and stirred for 15-30 min at ambient temperature.
The lipase beads were filtered through a medium porosity fritted glass
filter, and the polymers were washed with an additional 5-10 mL of
diethyl ether; the solvent was removed from the polymer by rotary
evaporation. Enzymes can form aggregates when combined with organic
solvents, and sonication is typically necessary to disrupt these ag-
gregates. Prolonged sonication of enzymes, however, can disrupt their
2
7
tertiary structure. Sonication carried out at a temperature below 30
2
8
°
C did not affect the catalytic activity of porcine lipase. When free
enzymes were used, they were combined with the hydroxypropyl-
terminated silicones and sonicated for 30-60 s at room temperature to
break apart enzyme aggregates. Carboxy-functionalized silicones were
then added to the free enzyme preparation and the reaction was
incubated at 35 °C for 72 h with stirring at 150 rpm. Reactions
employing free lipases were not conducted at 70 °C to avoid thermal
denaturation. Preparations in isooctane: Carboxy- and hydroxy-
functionalized silicones (1:1 ratio) were dissolved into 1 mL of
isooctane. Enzyme powders were suspended in 1 mL of isooctane,
sonicated for 30-60 s at room temperature, and combined with the
silicone solution. The reaction vessel was incubated at 35 °C for 72 h.
Reactions were terminated the same way as those containing im-
mobilized lipase. A complete list of enzymes, their specific activity,
and reaction temperatures are listed in Table 1.
9
as the acyl acceptors.
To alleviate these background rates of esterification and,
subsequently, gain a better understanding of the role of the
enzyme, two routes were examined. First, enzyme-free controls
were performed at a lower temperature and, second, 4 was used
as the acyl donor. Reactions carried out at 35 °C generated
minimal background polytransesterification in the range of from
0
-9%. Enzyme-free controls using 4 at 35 °C, 50 °C, and 70
°
C over 24 h did not result in any detectable polytransesterifi-
1
cation as determined by H NMR. Based on these results we
chose to work with 4 as the acyl donor for the remainder of the
project.
Results and Discussion
The design and synthesis of biocompatible silicone-derived
materials will benefit from expanding the diversity of available
CALB Loading. Lipase-mediated polymerizations have typi-
cally been performed employing 10% w/w of the immobilized

Biocatalytic Synthesis of Silicone Polyesters
Biomacromolecules, Vol. 11, No. 7, 2010 1821
corresponding to an average degree of polymerization (DPavg
)
of 4 and M (NMR) ) 1100 g/mol (Table 3). The byproduct
n
from these reactions was methanol, which can participate in
the reverse reaction to regenerate the starting diester. Without
the constant removal of methanol the synthesis of higher
molecular weight polymers would be be limited. An increase
in the temperature to 50 °C afforded a higher degree of monomer
conversion to 80% (DPavg ) 5, M
did an increase in temperature to 70 °C (89.2 ( 10.1% (DPavg
93, M (NMR) ) 2600 g/mol). The circled resonances in
n
(NMR) ) 1400 g/mol) as
)
n
Figure 3 represent the methylene protons on the carbon proximal
to the hydroxyl group before and after transesterification. The
1
3
C NMR spectrum supported a high degree of transesterification
as only one carbonyl resonance at 173.4 ppm, was clearly visible
29
(see Supporting Information). The Si NMR spectrum indicated
that none of the disiloxane bonds were hydrolyzed during the
course of the reaction. The FTIR spectrum was dominated by
a strong absorbance at 1737 cm representing the CdOstr of
Figure 2. Effect of increasing the amount of CALB that was loaded
into the reaction mixture. For the esterification of 4 with 1 there was
no significant change in the reaction rate beyond 10% CALB.
-
1
the polyester, but not the methyl ester that is normally located
-
1
at 1743 cm . Noticeably absent from the FTIR spectrum was
the OH stretching mode of the alcohol that nominally appears
lipase to the mass of the monomers. However, no expectation
was made that this would be the ideal loading for producing
silicone polyesters. In order to establish the ideal enzyme loading
for this system, the amount of CALB was altered over the course
of several experiments while keeping the concentration of the
monomers constant. All reactions were carried out at 70 °C as
-1
at ∼3300 cm .
When diol 2 was substituted for 1, polytransesterification with
4
1
was achieved with 73.8 ( 12.9% (DPavg ) 4, M
900 g/mol), 81.9 ( 2.9% (DPavg ) 6, M (NMR) ) 2700
(NMR) ) 3800 g/mol)
n
(NMR) )
n
g/mol), and 87.3 ( 7.0% (DPavg ) 8, M
n
9
monomer conversion at 35, 50, and 70 °C, respectively. At lower
temperatures the increase in the molecular weight of the silicone
diol led to minor decreases in the degree of esterification. These
differences were less pronounced at 50 and 70 °C, suggesting
that the molecular mass of the diol had little effect on the ability
of the enzyme to polymerize these chemical species. These
results are in contrast to those reported by Guo et al. where it
was observed that a higher degree of esterification could be
realized by using a higher molecular weight silicone diol or by
previously described. When 5 and 10% w/w of enzyme
compared to monomer mass was employed, the rate of poly-
transesterification increased by approximately 30%. The rate
constants were determined as described below (vide infra) and
yielded k
) 0.2733 h- to k10 ) 0.5497 h (where the subscript
1
-1
5
number is the weight percent of CALB used for the reaction;
Figure 2). This change did not result in any change in the
n
number average molecular weight (M ) of the silicone polyester.
In both instances, the average M was 1400 g/mol after 24 h of
n
12
using lower reaction temperatures. Their arguments were based
on thermal denaturation of the immobilized enzyme at higher
reaction temperatures. On the other hand, Poojari et al. increased
the weight average molecular weight of coblock silicone
incubation. At the highest enzyme loading, 20% CALB, the
reaction mixture became extremely viscous within the first hour
and the enzyme beads began to creep up the walls of the reaction
vessel. The enzyme beads were scraped back into the reaction
mixture every 30 min to maintain a more intimate contact
between the enzyme and the monomers. When 20% CALB was
used there was no significant change between the rate of reaction
9
polyesters by increasing the reaction temperature. We suspect
that the observed differences are attributable to the different
substrates and minor methodological modifications that were
employed in each study.
or the observed M
n
, over the 10% loading of CALB, k20
)
.6389 h-1 and M
Free lipases and proteases were screened as a secondary
component to this work to determine if they would also be
suitable candidates for mediating the polytransesterification of
functionalized silicone monomers. Type VI lipase from C.
rugosa, lipase from wheat germ, R-chymotrypsin, and papain
were screened as potential biocatalysts. Unfortunately, none of
these enzymes performed as desired, and as a result, wheat germ
lipase, R-chymotrypsin, and papain were removed from further
experimentation.
0
n
) 1800. Similar trends have been observed
9
,12
in other CALB-catalyzed copolymerizations.
As the con-
centration of CALB was increased, the corresponding increase
in reaction rate was not observed (i.e., a doubling of CALB
concentration did not result in a doubling of the reaction rate).
As a result of the decreasing returns to scale, 5% CALB was
chosen as the ideal catalyst loading for these experiments.
Lipase-Catalyzed Synthesis of Silicone Polyesters. Poly-
transesterification reactions of silicone-derived monomers were
examined using CALB catalysis over a range of reaction
temperatures (35, 50, and 70 °C). Low temperature reactions
It is known that LCR contains a tunnel near the active site
3
6
that can accommodate a fatty acid chain. However, we were
unsure if the small size of the tunnel near the active site, relative
to a dimethylsiloxy group, was inhibiting the processing of
silicone-based substrates. By reducing the local steric bulk near
the carbonyl group, it was believed that the free lipase would
be able to carry out the desired transformations. Carboxydecyl
polydimethylsiloxane (5; MW ) 1000 g/mol) contains an alkyl
spacer of nine methylene units between the carbonyl group and
the siloxane chain and as such more closely resembles the
natural fatty acid substrate that is typical for lipases. When LCR
was combined with 1 and 5 under solvent-free conditions,
(
35 °C) were carried out to allow for direct comparisons to be
made with free enzymes, which are apt to denature at elevated
temperatures. The success of these reactions, measured by the
percent of monomer converson, was analyzed using H NMR.
1
1
The H NMR resonance for the methylene protons proximal to
the hydroxyl group became shifted downfield from 3.60 to 4.02
ppm due to additional deshielding afforded by the esterified
carboxylate (Figure 3).
Initial results indicated that when CALB was charged with 4
and 1 at 35 °C, monomer conversion was low at 74.3 ( 12.9%

1
822 Biomacromolecules, Vol. 11, No. 7, 2010
Frampton et al.
1
(1H NMR) )
Figure 3. Representative H NMR spectra of 1 (top), 4 (middle) and a silicone polyester with >91% monomer conversion and M
n
∼
5500 g/mol. The circled resonances represent the methylene protons on the carbon alpha to the hydroxyl group before and after esterification.
a
n w
Table 3. Degree of Esterification, M , M , and PDI for Each Silicone Diol as Mediated by CALB
b
b
c
M+ (MS)d
e
f
g
diol
T (°C)
%
DPavg
M
n
(NMR)
M
n
(MS)
M
w
(MS)
PDI
3
3
HP-TMDS
35
74.3
80.0
89.2
73.8
81.9
87.3
4
5
9
4
5
8
1100
1400
1750
1900
2700
3800
1920
2661
1920
3250
3598
2940
975
1050
950
1300
1600
925
1100
1200
1000
1450
1900
1050
1.13
1.15
1.11
1.10
1.15
1.12
5
7
0
0
HP-PDMS
35
5
7
0
0
a
Typical reactions were incubated for 72 h with magnetic stirring. The presented data is the average of multiple trials for each reaction condition.
b
1
c
Percent conversion of silicone diol monomer from H NMR. Determined using the equation M
n
w
) DP(M of repeat unit/2) + 32, where DP ) 1/(1 -
1
d
p), where p ) the percent transesterification as determined from H NMR. The largest identifiable polymeric species from MALDI-ToF spectrum.
e
2
f
w
Determined using the equation M ) ΣAM ΣA, where A ) peak area and M ) fragment mass for all possible copolymeric fragments. Determined
g
n w n
using the equation M ) ΣAM/ΣA, where A ) peak area and M ) fragment mass for all possible copolymeric fragments. PDI ) M /M determined from
MALDI-ToF MS.
polytransesterification could not be detected by 1H NMR.
However, when the enzyme was suspended in isooctane,
incubation at 70 °C. To achieve polymers of higher molecular
weight, the reaction was continued for an additional 48 h with
intermittent vacuum application to prevent the build-up of
methanol. After this extended time period, polytransesterification
could routinely be achieved in >90% conversion.
approximately 64% (M
conversion could be realized after 72 h of incubation. By
comparison, CALB carried out the same transformation in 82%
n
(NMR) ) ∼1750 g/mol) monomer
(
M
n
(NMR) ) ∼3500 g/mol) at 35 °C, which could be improved
Polytransesterifications are generally assumed to be second-
3
7
to 93% (M
n
(NMR) ) ∼8950 g/mol) when the reaction
order reactions. This was subsequently shown to be the case
when using CALB as a catalyst by removing aliquots from the
temperature was increased to 70 °C. Enzyme-free controls were
performed, and in all cases there was no detectable polytrans-
esterification.
1
reaction mixture at fixed intervals followed by H NMR analysis.
1
Each H NMR spectrum was acquired by dissolving a 4 µL
Enzyme Esterification Reaction Kinetics. In the absence
of CALB, polytransesterification could not be detected by H
NMR even after 24 h of incubation at 70 °C (Figure 4); similar
trends were observed when enzyme-free controls were per-
formed at lower temperatures (data not shown). The polytrans-
esterification of 1 and 4 reached nearly 70% by 24 h of
3
aliquot from the reaction mixture into CDCl ; the initial
1
spectrum, representing time zero, was acquired prior to the
addition of CALB. The change in the integration of the reso-
nances associated with the newly forming ester were followed
as described (vide infra). A plot of x/a(a - x), where a represents
the amount of either 1 or 2 and x represents the amount of 1 or

Biocatalytic Synthesis of Silicone Polyesters
Biomacromolecules, Vol. 11, No. 7, 2010 1823
Table 4. Second-Order Rate Constants for CALB-Catalyzed
Polytransesterifications Determined from Line Fitting of
Experimental Data
temp (°C)
k
2
(hr-1)
R2
3
5
7
5
0
0
0.0719
0.1868
0.4279
0
0.9549
0.9972
0.9988
enzyme-free (70)
derived from 1,6-hexanediol and diethyl carbonate increased
6
over the temperature range of 60-90 °C.
When 2 was used as the acyl acceptor, the rate of reaction
was increased by approximately 10% over that for 1. After 24 h,
both reactions exhibited similar rates; however, at this point,
the rate of polytransesterification of 1 began to outpace that of
2 (Figure 6). The increased hydrophobicity of the longer silicone
chain appeared to have promoted the initial higher reaction rate.
This trend is supported by previous reports where it was
observed that increasing the hydrophobicity of the monomers
Figure 4. The polytransesterification of 1 and 4 under different
temperatures, 35 °C (red diamond), 50 °C (blue triangle) and 70 °C
(
black square) using CALB as catalyst. An enzyme-free control
reaction (green circle) was performed at 70 °C and did not result in
1
any polytransesterification, as determined by H NMR. Error bars
9
drove the reaction forward. This observation was contrary to
represent the standard error of the means of three replicates.
findings by Guo et al. in which the initial stages of the
esterification of lower molecular weight silicone diols proceeded
more readily due to increased mobility. Only during the later
stages of the reaction did the higher molecular weight silicone
2
consumed versus time (t) confirmed a linear relationship
between the variables. Kinetic analyses of N435-catalyzed
condensations of aminopropyl functionalized silicones with
adipic acid illustrated the increase in the DPavg as well as M as
n
1,12
diol give a higher degree of polymerization. It was postulated
that during the later stages of the reaction that monomer
hydrophobicity had a stronger effect on the reactions progress.
When the later stages of the reaction were compared for the
larger silicone diol, there was a reduction in the reaction rates
that was greater than that observed for the lower molecular
silicone diol. At 35 °C the change in the reaction rate went
1
0
a function of time.
Polytransesterifications were carried out at 35, 50, and 70
°
C. At each temperature, a plot of 1/(1 - p), where p is the
proportion of the silicone diol that has been converted to the
ester against time (t) for the initial stages of the reaction yielded
the second-order rate constant k
2
(Figure 5 and Table 4). The
-1
from 0.0663 to 0.0172 h (early stage to late stage) while at
later stages of the polytransesterification were analyzed to
determine how the reaction progressed over the long term. A
plot of 1/(1 - p) versus time showed an inflection point where
the reaction rate slowed by approximately 50%. It should be
noted that the reaction at 50 °C slowed by nearly 80% during
the late stages of the reaction. It is unclear why at this
temperature the reaction behaves differently than at 35 and 70
-1
5
0 °C the reaction rate changed from 0.124 to 0.0262 h . At
the highest reaction temperature there was no change in the
reaction rate between 24 and 72 h; the slope of the line was
essentially zero, suggesting the reaction had reached equilibrium.
The general dependence of the rate of a reaction on temper-
ature can be quantified using the Arrhenius equation (1).
°
C. This transition typically occurred between 60-80% mono-
-
Ea
mer conversion.
k ) Ae RT
(1)
Consistent with other examples of enzyme-mediated con-
densations, an increase in the temperature afforded higher
reaction rates. Jiang et al. showed that the molecular weight of
copolymers from the copolymerization of diethyl carbonate, a
diethylester and a diol increased as the reaction temperature
The activation energy, E
interpreted directly from a plot of ln k versus 1/T, which yields
a straight line with a slope of -E /R and an intercept of ln A.
When the Arrhenius equation is used, the activation energy,
, when 1 was the acyl acceptor was found to be 52.48 kJ/mol
a
, and the Arrhenius factor can be
a
increased from 60 to 80 °C but then decreased at 90 and 95
7
°C. Similarly, the molecular weight of polycarbonate materials
E
a
Figure 5. Time course plots of the changes in the average degree of polymerization for CALB-catalyzed polytransesterification of 3HP-TMDS
with CPr-TMDS-DME at 35 °C (red diamond), 50 °C (blue triangle), and 70 °C (green square). Error bars represent the standard error of the
means of replicate trials.

1
824 Biomacromolecules, Vol. 11, No. 7, 2010
Frampton et al.
silicone diol. The activation energy for the CALB-catalyzed
polytransesterifications was 52.48 kJ/mol and the Arrhenius
7
factor was 5.618 × 10 when 3HP-TMDS was the diol and
7
5
0.56 kJ/mol and 2.927 × 10 when 3HP-PDMS was the diol.
The lipase from C. rugosa catalyzed only the esterification
of carboxydecyl-functionalized polydimethylsiloxane and 3HP-
TMDS when an organic solvent was employed at 35 °C. Two
proteases, R-chymotrypsin and papain, were screened, but
neither performed any of the desired polymerizations.
Acknowledgment. The authors would like to thank T. Jones
(
Brock University) for acquisition of MALDI-TOF MS data.
Funding for this work was provided by the Ontario Partnership
for Innovation and Commercialization (OPIC) and the Ontario
Centre for Excellence (OCE). M.B.F. was supported by graduate
scholarships from OGS (2007-2008) and OGSST (2008-2009,
2009-2010).
Figure 6. Effect of increased chain length of silicone diols 1 and 2
on the degree of polytransesterification with 4. Increasing the chain
length from a disiloxane (blue circle) to a polysiloxane (red diamond)
led to a reduction in the rate of polytransesterification at 70 °C when
Supporting Information Available. Examples of spectra
obtained in the synthetic and kinetic analysis of the polymer
systems. This material is available free of charge via the Internet
at http://pubs.acs.org.
5
% w/w CALB was used as the catalyst. Error bars represent the
standard error of the means for replicate trials.
References and Notes
(
1) Stevens, M. P. Polymer Chemistry An Introduction, 3rd ed.; Oxford
University Press: New York, NY, 1999.
(
2) Brook, M. A. Silicon in Organic, Organometallic and Polymer
Chemistry; John Wiley and Sons: New York, 2000.
(
3) White, J. W.; Treadgold, R. C. Organofunctional Siloxanes. In Siloxane
Polymers; Clarson, S. J., Semlyen, J. A., Eds.; Prentice Hall:
Engelwood Cliffs, New Jersey, 1993.
(
4) Thomas, D. R. Cross-Linking of Polydimethylsiloxanes. In Siloxane
Polymers; Clarson, S. J., Semlyen, J. A. Eds.; Prentice Hall: Engelwood
Cliffs, New Jersey, 1993.
(
(
(
5) Bonduelle, C.; Martin-Vaca, B.; Bourissou, D. Biomacromolecules
2
009, 10, 3069–3073.
6) Jiang, Z.; Liu, C.; Xie, W.; Gross, R. A. Macromolecules 2007, 40,
934–7943.
7) Jiang, Z.; Liu, C.; Gross, R. A. Macromolecules 2008, 41, 4671–4680.
8) Zini, E.; Scandola, M.; Jiang, Z.; Liu, C.; Gross, R. A. Macromolecules
008, 41, 4681–4687.
7
(
2
(
9) Poojari, Y.; Palsule, A. S.; Cai, M.; Clarson, S. J.; Gross, R. A. Eur.
Polym. J. 2008, 44, 4139–4145.
Figure 7. Second-order rate constant vs the reaction temperature
for CALB-catalyzed polytransesterifications using 1 (red circle) and
(
(
(
(
10) Sharma, B.; Azim, A.; Azim, H.; Gross, R. A.; Zini, E.; Focarete,
M. L.; Scandola, M. Macromolecules 2007, 40, 7919–7927.
11) Mosurkal, R.; Samuelson, L. A.; Parmar, V. S.; Kumar, J.; Waterson,
A. C. Macromolecules 2007, 40, 7742–7745.
2
(blue diamond) as the acyl acceptor. Reactions were performed at
35, 50, and 70 °C using 5% w/w of CALB. Error bars represent the
standard deviation for replicate trials.
12) Guo, L.; Zhang, Z.; Zhu, Y.; Li, J.; Xie, Z. J. Appl. Polym. Sci. 2008,
1
08, 1901–1907.
13) Dai, S.; Xue, L.; Zinn, M.; Li, Z. Biomacromolecules 2009, 10, 3176–
and the Arrhenius factor was determined to be A ) 5.618 ×
3181.
7
1
0 (Figure 7). When the higher molecular weight silicone diol
(14) Poojari, Y.; Clarson, S. J. Silicon 2009, 1, 165–172.
(
15) Kato, M.; Toshima, K.; Matsumura, S. Biomacromolecules 2009, 10,
66–373.
2
was the acyl acceptor, the activation energy was 50.56 kJ/
3
7
mol and the Arrhenius factor was A ) 2.927 × 10 , demonstrat-
ing that the increase in the molecular weight of the silicone
diol had no effect on the temperature dependence of the
polymerization reactions.
(
16) Eriksson, M.; Fogelstr o¨ m, L.; Hult, K.; Malstr o¨ m, E.; Johansson, M.;
Trey, S.; Martinelle, M. Biomacromolecules 2009, 10, 3108–3113.
(17) Olsen, D. A.; Sheares, V. V. Macromolecules 2006, 39, 2808–2814.
(
(
18) Poojari, Y.; Clarson, S. J. Chem. Commun. 2009, 6834–6835.
19) Puskas, J. E.; Sen, M. Y.; Seo, K. S. J. Polym. Sci., Part A: Polym.
Chem. 2009, 47, 2959–2976.
(
20) Noritomi, H.; Nichida, S.; Kato, S. Biotechnol. Lett. 2007, 29, 1509–
Conclusions
1
512.
(21) Li, G.; Raman, K.; Xie, W.; Gross, R. A. Macromolecules 2008, 41,
003–7012.
The immobilized lipase from C. antarctica has been shown
to be a suitable enzyme for mediating the biological polytrans-
esterification of silicone-derived materials under solvent-free
conditions. The degree of esterification was dependent on the
molecular weight of the acyl donors/acceptors, the temperature
at which the reaction was carried out, and the amount of en-
zyme loaded into each reaction. Generally, there were positive
correlations between the degree of esterification with temper-
ature and enzyme loading. A negative correlation was found to
exist between the reaction rate and molecular weight of the
7
(22) Bassindale, A. R.; Brandstadt, K. F.; Lane, T. H.; Taylor, P. G. J. Inorg.
Biochem. 2003, 96, 401–406.
(23) Frampton, M.; Vawda, A.; Fletcher, J.; Zelisko, P. M. Chem. Commun.
2008, 5544–5546.
(24) Fukuoka, T.; Tachibana, Y.; Tonami, H.; Uyama, H.; Kobayasi, S.
Biomacromolecules 2002, 3, 768–774.
(
(
25) Uyama, H.; Fukuoka, T.; Komatsu, I.; Watanabe, T.; Kobayashi, S.
Biomacromolecules 2002, 3, 318–323.
26) Zhang, C.; Laine, R. M. J. Am. Chem. Soc. 2000, 122, 6979–6988.
¨
¨
(27) Ozbek, B.; Ulgen, K.O. Process Biochem. 2000, 35, 1037–1043.

Biocatalytic Synthesis of Silicone Polyesters
Biomacromolecules, Vol. 11, No. 7, 2010 1825
(
(
28) Goodman, L. P.; Dugan, L. R., Jr. Lipids 1970, 5, 362–365.
29) Brook, M. A.; Zelisko, P. M.; Walsh, M. J.; Crowley, J. N. Silicon
Chem. 2002, 1, 99–106.
(34) Ragheb, A. M.; Brook, M. A.; Hrynyk, M. Biomaterials 2005, 26
(14), 1653–1664.
(35) Klibanov, A. M. Trends Biochem. Sci. 1989, 14, 141–144.
(36) Cygler, M.; Schrag, J. D. Biochim. Biophys. Acta 1999, 1441, 205–
214.
(
30) Zelisko, P. M.; Brook, M. A. Langmuir 2002, 18 (23), 8982–8987.
31) Zelisko, P. M.; Lopez Aguilar, A.; Brook, M. A. Langmuir 2007, 23
(
(
7), 3620–3625.
(37) Allcock, H. R.; Lampe, F. W.; Mark, J. E. Contemporary Polymer
Chemistry, 3rd ed.; Pearson Education, Inc.: Upper Saddle River, NJ,
1981.
(
32) Zelisko, P. M.; Flora, K. K.; Brennan, J. D.; Brook, M. A. Biomac-
romolecules. 2008, 9, 2153–2161.
(
33) Ragheb, A. M.; Brook, M. A.; Hrynyk, M. Chem. Commun. 2003,
2
314–2315.
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