Chemo-enzymatic pathways toward pinene-based renewable materials

Article abstract of DOI:10.1039/c9gc00718k

Sobrerol methacrylate (SobMA) was synthesized and subsequently polymerized using different chemical and enzymatic routes. Sobrerol was enzymatically converted from α-pinene in a small model scale by a Cytochrome P450 mutant from Bacillus megaterium. Conve

Full text of DOI:10.1039/c9gc00718k

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Title: Chemo-enzymatic pathways toward pinene-based renewable materials
a,c
a
a,c
a,b
a,c
a,b
A. Stamm , M. Tengdelius , B. Schmidt , J. Engström, P.O. Syrén , L. Fogelström and E.
a,b
Malmström
KTH Royal Institute of Technology, School of Engineering Sciences in Chemistry,
a
Biotechnology and Health, Department of Fibre and Polymer Technology, Division of
b
Coating Technology, Wallenberg Wood Science Center, Teknikringen 56-58, SE-100
c
4
4 Stockholm, Sweden SciLifeLab, Division of Protein Technology, Tomtebodavägen
3, Box 1031, SE-171 21 Solna, Sweden
2
Graphical Abstract:
Abstract
Sobrerol methacrylate (SobMA) was synthesized and subsequently polymerized using different
chemical and enzymatic routes. Sobrerol was enzymatically converted from -pinene in a small
model scale by a Cytochrome P450 mutant from Bacillus megaterium. Conversion of sobrerol
into SobMA was performed using both classical ester synthesis, i.e., acid chloride-reactions in
organic solvents, and a more green approach, the benign lipase catalysis. Sobrerol was
1

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successfully esterified, leaving the tertiary alcohol and “ene” to be used for further chemistry.
SobMA was polymerized into PSobMA using different radical polymerization techniques,
including free radical (FR), controlled procedures (Reversible Addition Fragmentation chain-
Transfer polymerization, (RAFT) and Atom Transfer Radical Polymerization (ATRP)) as well as
by enzyme catalysis (horseradish peroxidase-mediated free radical polymerization). The resulting
o
polymers showed high glass-transition temperatures (Tg) around 150 C, and a thermal
o
degradation onset above 200 C. It was demonstred that the Tg could be tailored by
copolymerizing SobMa with appropriate methacrylate monomers and that the Flory-Fox equation
could be used to predict the Tg. The versatility of PSobMA was further demonstrated by forming
crosslinked thin films, either using the ‘ene-functionality for photochemically initiated ‘thiol-
ene’-chemistry, or reacting the tertiary hydroxyl-group with hexamethoxymethylmelamine, as
readily used for thermally curing coatings systems.
INTRODUCTION
Reducing the need for petroleum-based materials is one of the main challenges in the
contemporary society. Proposed practical solutions to this are either to reduce the use of fossil-
based consumer products, or to develop alternative materials originating from renewable sources.
Most likely, a combination of both strategies is required to accelerate the transition towards a
more sustainable society. Lately, the members of the European Parliament voted for the ban of
single-use plastic by 2021, increasing the demand for new materials.1
Still, the vast majority of all plastics produced are based on fossil-sources; only around one
2
percent of the about 300 million tons of plastic produced annually is bio-based. There are
different methods for obtaining renewable polymers. One common strategy is to utilize abundant
biopolymers available in biomass, mainly cellulose, and by degradation/refining produce
2

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renewable equivalents in order to replace monomers traditionally obtained from fossil-sources,
e.g., ethylene and propylene. One major drawback with this is that the biopolymer has to be
degraded into small molecules and then again polymerized to large molecules. Furthermore, the
production of the polymers utilizes the same harsh industrial processes regardless of the origin of
3
the monomers. Another strategy is to use biopolymers originating from renewable sources, such
as cellulose, lignin, starch and chitosan, in their inherent state to produce sustainable materials.3,4
This approach is hampered by the fact that biopolymers are associated with large property
variations as an effect of varying growth conditions (growth season, locus etc.), significantly
challenging their industrial utilization. A third strategy is to use novel renewable monomers from
biomass, not readily available from fossil sources, to produce renewable polymers with properties
comparable to fossil-based counterparts.
Trees and plants have proven particularly useful as sources for sustainable monomers.3,5,6 Besides
containing significant amounts of biopolymers, which can be used either directly for producing
4
,6
materials or to produce monomers by depolymerization followed by fermentation and/or
5
upgrading, these sources are also rich in small molecules useful as monomers, such as furans,
3
,5
fatty acids, and terpenes. With the rapid emergence of modern biorefineries, focusing not only
on energy recovery, an array of waste-stream products has become more available and, therefore,
7
more interesting from a commercial point of view. Terpenes belong to one such group of
compounds that are rendering increasing interest in this context. Terpenes constitute one of the
most diversified classes of natural products with essential functions in a broad range of
8
,9
organisms. Terpenes/terpenoids have, for instance, found important applications, or potential
1
0
11
12
applications, in areas such as fragrances and flavors, biofuels, fine chemical synthons, and as
8
anticancer and antiviral agents. Terpenes are assembled from the universal C -building blocks,
5
dimethylallyl diphosphate and isopententyl diphosphate, to yield linear polyisoprene building
3

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3
blocks . The extensive structural variations found in terpenes is an effect of enzyme-catalyzed
cyclizations to result in chiral and multicyclic natural products, which presents further
1
2
opportunities for producing polymers with a wide range of properties beyond natural rubber
(i.e. polyisoprene). The main source of terpenes is the pulp industry-derived pine-tree resin
3
turpentine, industrially available on ca. 300,000 ton scale annually. The primary terpene in
6
turpentine is α-pinene. It has a bicyclic unsaturated structure (Scheme 1), and has been reported
to result in polymers with alternating linear and cyclic units via cationic polymerization.13–15 Still,
achieving controlled polymerization of pinene monomers remains a challenge, which has spurred
an increased interest towards unlocking their inherent potential by activation through oxidation
by chemical and biocatalytic methods.16–18 Biocatalysis displays a great potential to replace harsh
chemical synthesis with green options for the sustainable generation of medicines, biochemicals
1
9
and materials. In particular, by capitalizing on synthetic-biology approaches, mild oxidations
required for monomer activation – which would be challenging to access by traditional organic
2
0
21
synthesis – can be achieved in water with oxygen as co-substrate. Over the last years, many
studies have been devoted to the use of enzymes as mediators for different polymerization
techniques.22–26 Polymers from terpenes, or chemically modified terpenes, exhibit a wide range of
molecular weights and thermal properties depending on the terpene structure/composition and
polymerization technique.12,27 In the pursuit of novel polymeric materials, exploring the toolbox
of terpene-based polymers holds great potential.28
One promising underutilized terpene is sobrerol, an unsaturated diol, Scheme 1, which is formed
2
1
directly via biotransformation of α-pinene in the fungi Armillaria mella or through aqueous
2
9
30
31
oxidation. Sobrerol has shown antimycotic properties, to prevent pulmonary hypertension
and the development of breast cancer.32
4

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The multifunctional structure of sobrerol presents several opportunities for producing polymers.
Its cyclic structure furthermore holds great promise for producing higher T -materials, in contrast
g
to the majority of available green monomers that predominantly have non-cyclic structures.
However, despite its functional groups, offering versatility in both polymer production and post-
polymerization modifications, sobrerol is yet to be further explored as a novel monomer for
polymeric materials.33
Structurally similar terpenes and terpenoids have been converted to polycarbonates,34–36
3
7
38
17
27
polyethers, polyesters, polyamides, and poly(meth)acrylates upon modification. The diol
present in sobrerol presents opportunities for producing polymers of all the aforementioned
classes. (Meth)acrylate polymers are widely used in the automotive, paint, home electronics, and
medical-device industries, with a steadily increasing global market.39,40 Due to these widespread
industrial applications, methods for large-scale production of (meth)acrylates are becoming
4
1
increasingly more sustainable. The industrial usage and well-developed polymerization
4
2
techniques of methacrylates, make sobrerol-based methacrylate polymers an interesting starting
point for studying the potential of sobrerol as a renewable component in materials. Fonseca et al.
recently published the use of SobMA as a substituent for polystyrene in the formation of
polyester resins. 33 Sobrerol was obtained from -pinene oxide in the presence of CO and, after
2
(meth)acrylation, incorporated into unsaturated polyester-resin formulations as a reactive diluent.
The sobrerol-based formulations exhibited similar properties to those of the styrene-based
polyester resins while also improving the environmental compatibility due to its biobased origin,
low volatility and thus, a reduced exposure of VOC’s to humans and the environment.
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In this work, we have developed a chemoenzymatic “green” route for the synthesis of SobMA.
The polymerization of SobMA was studied along with the properties of the resulting polymers.
We present different routes to synthesize sobrerol-based polymethacrylates (PSobMAs). The
o
resulting polymethacrylates showed a T around 150 C. It was possible to tailor the T by
g
g
copolymerization of various methacrylate monomers. The great versatility of PSobMA allows
different material applications, e.g., coatings, and was demonstrated by bsubsequent crosslinking
of the functional side-groups, using either thiol-ene or condensation coating chemistry.
ATRP/ RAFT/
Free radical
polymerization
OH
methacryloyl chloride,
TEA, DMAP
O
O
O
O
P450-BM3/2M
O , H O
DMF or 2-MeTHF
OH
2
2
OH
OH
HRP Typ II
THF, H2O
Lipase
Vinyl methacrylate
(-)--Pinene
Sobrerol
Sobreryl methacrylate
Poly(sobreryl methacrylate)
(
SobMA)
(PSobMA)
I
II
III
IV
Scheme 1. Chemoenzymatic pathways from (-)α-pinene to PSobMA. I-II: Conversion of α-
pinene into SobMA catalyzed by Cytochrome P450 from Bacillus megaterium
(CYP102A1)A74G/L188Q mutant (P450-BM3/2M), cofactors have been omitted for clarity. II-
IV: Enzymatic conversion or chemical synthesis of SobMA followed by radical polymerization
through different polymerization techniques.
RESULTS & DISCUSSION
Biotransformation of (-)-α-pinene to prepare trans-sobrerol. The ability of Cytochrome P450
monooxygenases to, under mild conditions, insert oxygen at activated positions (eg allylic)
6

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makes them a promising class of biocatalysts for oxidation of α-pinene and other monoterpenes
into possible monomers.43–46 To afford trans-sobrerol from (-)-α-pinene in one-pot via the
corresponding epoxide, the biotransformation was performed in vitro using purified P450-
BM3/2M adopted to procedures described for glucose-6-phosphate (G6P), and G6P
dehydrogenase (G6PDH) was used for the co-factor regeneration (i.e. adenine dinucleotide
phosphate NADPH) (Figure S 1).46,47 After 22 h reaction time under aqueous conditions, 42 %
conversion (according to GC/MS) of (-)-α-pinene into trans-sobrerol was observed without
intermediate work-up, which is in good agreement with recently reported data (Figures S 2 and S
4
6
3
). At this point, this reaction was demonstrated on a small scale, in support of previous
4
6
reports why all further experiments were conducted on commercially available trans-sobrerol.
Chemical transformation of trans-sobrerol to SobMA. The SobMA was synthesized via a
conventional chemical approach starting from commercially available sobrerol, using
methacryloyl chloride and triethyl amine in DMF. DMF was not a preferred solvent but despite
previous reports,48,49 sobrerol proved difficult to dissolve. Purification via medium pressure liquid
chromatography (MPLC) yielded the liquid SobMA monomer in 66 % yield. The monomer was
1
sensitive to self-polymerization and was therefore stored in the mobile phase after MPLC. H
NMR spectrum of SobMA showed a downfield shift of the signal corresponding to the methine
proton neighboring the secondary alcohol (signal a in Figure 1a-b) compared with sobrerol,
corroborating the successful ester formation. This signal and the signals corresponding to the ene-
protons in the ring and the methacrylate alkenes (peak e, n , and n in Figure 1b) were found to
1
2
have a 1:1:1:1 integral ratio. This confirmed that the monomer was selectively mono-
methacrylated. The signal of the methyl group on the ring (signal g in Figure 1 a and b) showed
an upfield shift compared with sobrerol, confirming a change in electron density in the six-
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1
3
membered ring. C NMR also displayed a noticeable downfield shift for the signal
corresponding to the carbon bound to the secondary alcohol (signal a in Figure 1Error!
Reference source not found. c,d and e), which further confirmed esterification of the secondary
alcohol. GC-MS and GC-FID (Figures S 4 - 5) confirmed methacrylation at the secondary
alcohol. A previous study showed that the tertiary alcohol in sobrerol, upon ionization in the mass
4
7
detector, dehydrates selectively to form a mass fragment 18 a.u. below its molecular ion. The
mass fragment at m/z = 220 in the monomer mass spectrum thus confirmed that the
methacrylation occurred selectively at the secondary alcohol, as this fragment is exactly 18 below
the molecular ion of the desired monomer. These combined results show the predominant
formation of a mono-methacrylated product with clear selectivity for the secondary alcohol over
the tertiary, during the methacrylation. This could prove useful in industrial applications as it
eliminates the need for protection/deprotection schemes.
8

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1
1
Figure 1. NMR spectra in CDCl . (a) H NMR spectrum of sobrerol. (b) H NMR spectrum of
3
1
3
13
SobMA. (c) C NMR spectrum of sobrerol. (d) C NMR spectrum of SobMA.
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One obvious drawback with the described synthesis is the use of DMF, which is a non-renewable
solvent and associated with reproduction toxicity. A renewable and less toxic solvent is hence
desirable, particularly upon scale-up. 2-Methyltetrahydrofuran (2-MeTHF) and is derived from
5
0
51
biomass and exhibit a low toxicity. Using identical conditions as described above, but 2-
MeTHF instead of DMF afforded SobMA in 57 % yield.
Enzymatic transformation of trans-sobrerol to SobMA. In our strive to design a ‘green
chemistry-approach’ with no use of CMR-classified chemicals, we demonstrated that it is
possible to accomplish SobMA from trans-Sob and vinyl methacrylate (VMA) using the enzyme
Amano lipase from Pseudomonas Fluorescenc (P. fluorescenc) at a high conversion (96%) and
without the need for purification. The conditions were adopted from Chen et. al., who reported
5
2
high conversions for the lipase-catalyzed acetylation of sobrerol. The highest conversion was
obtained at 80 °C and a molar ratio of Sob:VMA equal to1:56. However, it was found that high
conversions (~90%) were possible to achieve at a lower excess of VMA (1:20) (Error!
Reference source not found.Table S 1, Table S 2). Nevertheless, it should be mentioned that in
scales above 4g of sobrerol purification by MPLC had to be conducted to isolate the pure
product.
RAFT polymerization of SobMA. SobMA was polymerized by reversible addition-
fragmentation chain-transfer (RAFT) polymerization to assess its applicability as a methacrylate
monomer. SobMA was stored in a mixture of dichloromethane and ethyl acetate of which none is
suitable for thermally initiated free radical polymerizations. The solution volume was therefore
carefully reduced to prevent premature polymerization, whereafter the monomer was diluted by
DMF and polymerized using 4,4’-azobis(4-cyanovaleric acid) (ACVA) as the initiator and 4-
cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPAD) as the RAFT agent. The monomer
1
0

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1
conversion was followed by H NMR (Figure S 6) and the polymerization was confirmed by the
disappearance of the methacryloyl alkene signals at  = 6.07 and 5.51 ppm, as well as the upfield
shift of the signal corresponding to the proton neighboring the methacrylate group from 5.29 ppm
to 5.00 ppm. The ratio between the latter signals was used to determine the monomer conversion.
The monomer consumption over time followed the first-order kinetics (Figure S 7). This, in
combination with the linear evolution of molecular weight with monomer conversion
(Figure S 8), corroborated a reasonably controlled RAFT polymerization. After 8 hours at 70 °C
and conversions reaching above 80 %, the polymer was precipitated in diethyl ether and isolated,
yielding poly(sobreryl methacrylate) (PSobMA ) as a pinkish powder.
R
Three different RAFT polymerizations were performed, using three different [SobMA]:[CPAD]
ratios; 50:1, 100:1, and 200:1. The resulting polymers PSobMA , PSobMAR100, and
R50
1
13
PSobMAR200 were analyzed by NMR and SEC. Typical H NMR and C NMR spectra for
RAFT-derived PSobMAs are displayed in Error! Reference source not found.. The molecular
weights (M ) determined by SEC (Figure 3a) corresponded fairly well with the theoretical M
n
n
(Table 1). The dispersities Ð were all reasonably narrow. By varying the ratio of monomer to
RAFT agent it was possible to control the molecular weight.
FR and ATRP polymerization of SobMA. To further elaborate the potential as methacrylate
monomer, SobMA was polymerized using free radical (FR)- and atom transfer radical
polymerization (ATRP) techniques. As for the RAFT polymerizations, the ATRP reactions were
performed using three different [SobMA]:[HMTETA]:[EBrP]:[CuCl] ratios; 50:2:1:1, 100:2:1:1,
and 200:2:1:1. In the FR polymerizations, similar conditions were used as in the RAFT
polymerization but without the RAFT agent and with a reduced polymerization time (5 hours).
All polymers were analyzed by NMR and SEC to determine molecular weights and dispersity
1
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(Figure S 7 - 10). The results (Table 1) were reasonable for free-radical and ATRP
polymerizations and corroborated that the polymer chain length can be tailored using ATRP, as
observed for the RAFT polymerizations.
1
13
Figure 2. H (a) and C (b) NMR spectra in CDCl of PSobMA via RAFT polymerization.
3
1
2

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Table 1. Characterization of sobreryl methacrylate polymers.
Polymera
Method Ratio (Method)
Time
h)
Conv.b
(%)
91
M
c
M
d
Ðd
T
g
e
T
d
f
n, theo.
n, SEC
(
(g/mol) (g/mol)
48 200 2.4
11 000 7 200 1.2
(oC)
154
116
145
155
141
n.d.
133
74
(oC)
238
191
224
238
227
200
224
213
195
215
220
224
235
196
PSobMAFR
FR
250:1 (FR)
5
8
8
8
-
PSobMAR50
RAFT
RAFT
RAFT
RAFT
RAFT
RAFT
RAFT
RAFT
ATRP
ATRP
ATRP
Enz.
50:1:0.2 (R)
100:1:0.2 (R)
90
PSobMAR100
83
20 100 20 900 1.3
38 900 31 600 1.2
PSobMAR200
200:1:0.2 (R)
81
g
95/66g 29 500 28 000 1.2
88/70g 31 200 27 000 1.1
73/74g 25 100 27 800 1.2
67/81g 27 800 25 800 1.1
P(SobMAR100-b-MMAR100
)
(100:100) :1:0.2 (R) 24
g
P(SobMAR100-b-BMAR100
P(SobMAR100-st-MMAR100
P(SobMAR100-st-BMAR100
)
(100:100) :1:0.2 (R) 24
g
)
(100:100) :1:0.2 (R)
8
8
8
7
9
9
1
g
)
(100:100) :1:0.2 (R)
g
P(SobMAR25-st-BMAR75
PSobMAA50
)
(25:75) :1:0.2 (R)
39/48
75
13000
9 100
11000
1.2
41
50:2:1:1 (A)
100:2:1:1 (A)
200:2:1:1 (A)
42:1:1.48 (E)
10 500 1.2
132
138
141
149
37
PSobMAA100
62
15 000 16 900 1.2
34 500 20 900 1.2
PSobMAA200
71
PSobMA
E
89
n.d.
n.d.
39 800 1.9
51 100 2.3
P(SobMAE25-st-BMAE75
)
Enz.
(10.5:31.5):1:148 (E) 3
59/79
aThe the numbers refer to the target DP; the letter in the annotation refers to the polymerization
method where R = RAFT (ratio: [M]:[CPAD]:[ACVA]), FR = free-radical (ratio: [M]:[ACVA]),
A = ATRP (ratio: [M]:[HMTETA]:[EBrP]:[CuCl]) and E=enzymatic (ratio: [M]:[ 2,4-
b
pentanedione]:[H O ] (Oxidase ) 80 mg/mL (2.9 mg enzyme per mmol monomer)). Determined
2
2
1
c
d
by H-NMR. Calculated according to the RAFT and ATRP mechanisms. Determined by SEC
e
using DMF as eluent and PMMA standards. Determined by DSC using second heating to assess
f
the glass transition temperature. Determined by TGA at the temperature corresponding to 5 %
g
weight loss. Left figure refers to SobMA, right figure refers to MMA or BMA.
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Figure 3. Molecular weight distributions for poly(SobMA) synthesized using (a) RAFT and (b)
enzymatic polymerization.
Copolymerization of SobMA with MMA and BMA. To confirm the controlled character of the
RAFT polymerizations, block copolymers were synthesized using SobMA and methyl
methacrylate (MMA) or butyl methacrylate (BMA). SobMA was polymerized for 6 hours, under
the same conditions as the other RAFT polymers, reaching ca 80 % conversion of SobMA as
1
assessed by H NMR. Therafter, one molar equivalent of the co-monomer was added and the
1
polymerization was left for another 18h. H NMR samples acquired after a total of 24 hof
polymerization showed high monomer conversions, Table 1.The MMA conversion was
determined through the ratio between the signals corresponding to the methyl side groups, which
show an upfield move in shift from  = 3.69 ppm to  = 3.54 ppm upon polymerization
(Figure S 11). Similarly, the methylene protons neighboring the alcohol part of the methacrylate
ester in BMA showed an upfield shift from  = 4.14 ppm to  = 3.94 upon polymerization. The
ratio between these signals was consequently used to determine the BMA conversion
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(
Figure S 12). SEC analysis shows increasing M after the copolymerizations and retained
n
relatively narrow polydispersity (Figure S 13 - 14). This confirmed the chain extension of the
polymer chains, and thus the successful block formation. Analysis of the resulting P(SobMAR100
-
b-MMAR100) and P(SobMAR100-b-BMAR100), by SEC (Figure S 13 - 14), again showed molecular
weights comparable to the theoretical M (Table 1). Comonomer molar fractions of the isolated
n
polymers were [SobMA]:[MMA] = 1:0.61 and [SobMA]:[BMA] = 1:1.37 for P(SobMAR100-b-
1
MMAR100) and P(SobMAR100-b-BMAR100), respectively, as determined by H NMR (Figure S
1
5 - 16).
The possibility of tailoring the properties of the polymer materials by statistical co-
polymerization of SobMA with MMA or BMA was explored. Both monomers were added just
prior to the polymerization, which was left to proceed for 24 h under conditions similar as for the
homopolymers by RAFT. After 8 hours, monomer conversions of 73 and 74 %, for SobMA and
1
MMA respectively, and 67 and 81 %, for SobMA and BMA respectively, were detected by H
NMR. SEC analysis for the resulting P(SobMAR100-st-MMAR100) and P(SobMAR100-st-BMAR100
)
again showed molecular weights in good agreement with the theoretical molecular weights
(Table 1). The copolymer compositions were [SobMA]:[MMA] = 1:0.99 and [SobMA]:[BMA] =
1
:1.32 for P(SobMAR100-st-MMAR100) and P(SobMAR100-st-BMAR100), respectively, as
1
determined by H NMR (Figure S 17 - 18). The FTIR spectra of the four copolymers (block and
statistical) showed absorbance patterns similar to those of the homo RAFT polymers (Figure
S 19).
Enzymatic polymerization of SobMA. In addition to traditional radical polymerization
techniques, SobMA was also polymerized using horseradish peroxidase (HRP)53,54 in the presence
of low concentrations of hydrogen peroxide and acetylacetone as radical initiator, following the
1
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5
5
procedure described by Gross et al. SobMA was homo- and copolymerized with BMA at room
temperature in a 1:3 water:THF mixture. The reason why THF was used instead of 2-MeTHF was
the low miscibility of 2-MeTHF in water. The reaction time was 1 h for the homopolymerizations
1
and 3 h for the copolymerizations, respectively. The monomer conversion was determined by H
NMR and found to be above 80% for both homo- and copolymerization. After precipitation, the
resulting polymers were characterized by NMR and SEC (Figures 3b and S 20 - 22). The obtained
polymers were of high molecular weights, >45 kg/mol, and Ð~ 2.2, in good agreement as reported
by Gross et al. for PMMA.
Thermal Characterization of PolySobMA. To assess the potential material applications of
PSobMA, all polymers were analyzed by thermogravimetric analysis (TGA) and differential
scanning calorimetry (DSC). TGA analysis showed that all polymers undergo a three-step
degradation (Figures 4 and S 23). This multistep-degradation profile is known for
polymethacrylates and many studies were conducted to investigate and simulate the thermal
degradation profile of polymers like PMMA.56–58 The weight ratios between the observed
degradation steps correlate to the ratio of different chain-termination mechanisms (coupling or
disproportionation) and therefore slightly varies from one polymerization technique to another.
The degradation occurring at the lowest temperature (165 °C) is attributed to degradation through
5
8
chain-scissioning of the weak head-to-head linkages. The second step (around 270 °C) is
referred to as β-scissioning of the unsaturated chain ends formed during disproportionation and
finally, the last step is random scissioning within the remaining polymer chains. A study by
Yamago et. al. on the effect of reaction temperature on the ratio of combination and
disproportionation, showed an increased amount of combination at higher temperature. However,
4
59
the ratio became constant for polymers with Mn above 10 . This dependency is in good
agreement with our results and explains the larger weight drop for PSobMAR50 at 165 °C
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(reaction temperature 70 °C→ increased amount of coupling), as well as the higher magnitude of
the second degradation step for PSobMA (reaction temperature r.t. → increased amount of
E
disproportionation). The degradation profiles of the prepared statistical and block copolymers
generally show a decrased magnitude of the second degradation step which is in good agreement
5
8
to the degradation profiles of PMMA analyzed by Ferriol et. al. The degradation temperatures
(Td, defined at 5 % weight loss) of all polymers are found in Table 1.
100
PSobMAR50
PSobMAR100
PSobMAR200
80
60
40
20
0
PSobMAE
100
200
300
400
500
600
700
T (°C)
Figure 4. Thermograms (TGA) for enzymatic PSoBMA and RAFT PSobMAs of different chain
o
lengths at a heating rate of 10 C/min.
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DSC analyses (Figure S 24) showed that all homopolymers exhibited glass transition
o
temperatures (T ) between 110-155 C (see Table 1), with the maximum T for the polymer with
g
g
M = 30 000 g/mol. The statistical copolymer containing MMA showed a T comparable to those
n
g
of the homopolymers, whereas the statistical incorporation of BMA into the polymer structure
o
resulted in a markedly decreased T , close to 37 C (P(SobMA -st-BMA )). As expected, no
g
E25
E75
melting transitions could be detected for any of the polymers.
Coating preparation. In PSobMA, each repeating unit contains both a tertiary alcohol and a
substituted alkene. These groups render possibilities for versatile post-polymerization
modifications, e.g., crosslinking to form gels or coatings (Error! Reference source not
found.5). Crosslinking via alkenes can be conducted using thiol-ene chemistry, employing a
multi-functional thiol, even though similar cyclic alkenes are reported to react relatively slowly.60
To assess the possibility of utilizing the double bond (the “ene”) in the side-chain for for
crosslinking purposes, PSobMAs were mixed with trimethylolpropane tris(3-
mercaptopropionate) (TMTP, a trifunctional thiol), and a radical initiator (Irgacure 651). The
mixture was applied on a teflon substrate and cured under UV-irradiation. The resulting
crosslinked films were analyzed by FT Raman spectroscopy which showed reductions in the
-1
“
ene”-band, compared with PSobMAR50 (CH=C: 1678 cm , normalized to the carbonyl peak at
-1
1
727 cm ; Figure S 25 - 26Error! Reference source not found.). The successful crosslinking of
PSobMAs-films was further confirmed as films were insoluble in both THF and
dichloromethane, two solvents which readily dissolve PSobMAs (Figure S 27). A control
experiment was conducted to further verify the observations, the film was then irradiation in the
presence of the photoinitiator but in the absence of TMTP and the resulting film was readily
dissolved in both THF and dichloromethane. Further attempts to crosslink PSobMA were made
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via transetherifications, utilizing the tertiary alcohol rather than the alkene. PSobMAR50 and
P(SobMA -st-BMA ) were mixed with hexamethoxymethylmelamine (HMMM), a
E25
E75
commonly used crosslinker in thermally curable industrial coating applications. and subsequently
p-toluenesulfonic acid (PTSA) dissolved in a small amount of ethyl acetate was added. The
o
coating was applied onto a Teflon substrate andand cured at 140 C to a solid film. A slight
reduction in the band corresponding to the alcohol was observed by FTIR (Figure S 28),
indicating that crosslinking had occurred. The successful crosslinking were further verified by the
fact that the cured coating resins were insoluble (Figure S 29).
Figure 5. Crosslinking of functional side-chain groups on poly(sobreryl methacrylate) via thiol-
ene chemistry (left) and transetherification (right).
Conclusions
Sobrerol can be readily obtained by upgrading of α-pinene. Sobrerol can be transformed into
SobMA either by classical esterification, or by enzyme catalysis, and both methods show very
good selectivity for the secondary alcohol over the tertiary. SobMA can readily be polymerized
by RAFT, FR, and ATRP in DMF, 2-MeTHF, and THF, respectively. In contrast to the chemical
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catalysis, enzyme-mediated polymerization was performed in aqueous systems at room
temperature showing superior activity. Copolymers could be obtained with properties as expected
o
from stoichiometry. The synthesized polymethacrylates showed T s around 150 C and narrow
g
dispersities in the case of RAFT and ATRP. Tailoring of T was demonstrated by
g
copolymerization of methacrylate monomers. Further functionalization possibilities were
demonstrated by utilizing the remaining functional groups in PSobMA; thin films were
photochemically cured in the presence of multifunctional thiols or thermally cured using
HMMM. Our work demonstrates the paramount potential of upcycling underutilized biobased
building blocks into sustainable materials through chemo-enzymatic catalysis.
MATERIALS & METHODS
Materials. All chemicals were obtained from Sigma-Aldrich unless otherwise noted. Methyl
methacrylate (MMA) was passed through a column with Al O (activated, neutral, Brockmann
2
3
Activity I) before used to remove the MEHQ inhibitor.
Instrumentation. GC-MS (Shimadzu, GCMS-QP2010 Ultra) was performed on an Rxi®-5ms
30 m, 0.25 mm [inner diameter], 0.25 µm [film thickness], RESTEK). The temperature program
was set at 70 °C, before increasing to 300 °C with a rate of 20 °C/min and finally increased to
50 °C with a rate of 5 °C/min before holding at 350 °C for 10 min. For MPLC, a Biotage Isolera
Four system equipped with a UV detector and Biotage KP-Sil SNAP Cartridge columns was
(
3
1
13
used. H (400 MHz) and C (100 MHz) NMR spectra were recorded with a Bruker Avance AM
6
1
4
00 instrument. The signal of the deuterated solvent CDCl ( = 7.26 ppm ) was used as
3
reference. For SEC, a TOSOH EcoSEC HLC-8320GPC system was used equipped with an
EcoSEC RI detector and three PSS PFG 5 μm columns (microguard, 100 Å, and 300 Å).
Poly(methyl methacrylate) (PMMA) standards were used for calibration and toluene was used as
2
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internal standard. DSC was performed using a Mettler Toledo DSC 820 module. Samples (5-10
mg) were prepared in 100 μL aluminum crucibles. The samples were subjected to heating from
o
o
o
o
o
3
0 to 170 C (or 160 C), then cooled to -60 C, and then heated again to 170 C (or 160 C) at a
o
heating/cooling rate of 10 C/min under nitrogen flow (50 mL/min). The data obtained from the
second heating was used for analyses. For TGA, a Mettler Toledo TGA/DSC1 instrument was
o
used. Samples (5-7 μg) were prepared in 70 μL alumina crucibles and heated from 40 to 700 C
o
at a heating rate of 10 C/min under a nitrogen flow (50 mL/min). FTIR was performed on a
Perkin-Elmer Spectrum 100 FT-IR instrument equipped with a single reflection ATR system and
a MIR-TGS detector using a MKII Golden Gate (Graseby Specac Ltd, Kent, England). Spectra
-1
-1
were recorded over the range 4000-600 cm and based on 16 scans at an average 4.0 cm
resolution.
Sequences and strains for producing P450-BM3/2M. The vector with P450-BM3 gene inserted
into pET28a(+) plasmid was generously provided by Dr. Bettina Nestl, University of Stuttgart,
Germany. Site directed mutagenesis to afford the P450-BM3/A74G/L188Q (P450-BM3/2M) was
carried out following the Agilent Pfu Ultra II protocol with primers listed in Table S 3 with the
temperature program according to Table S 4. Competent E. coli BL21(DE3) [genotype: F–
ompT hsdSB(rB-, mB-) gal dcm (DE3)] were transformed with P450-BM3/2M_pET28a(+)
vector by heat-shock and the cells were plated on LB-agar plate [1.6% (w/v) tryptone, 0.5% (w/v)
yeast extract, 1% (w/v) NaCl, 1% (w/v) agar] supplemented with 50 mg/L kanamycin.
Protein expression and purification. Enzyme was produced by inoculating 25 ml 2xYT media
[1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl] supplemented with 50 mg/L
kanamycin (2xYT-Kan media) with a single colony from the transformed BL21(DE3) cells and
incubating overnight at 37 °C, 200 r.p.m. Overnight culture was transferred to 600 ml of 2xYT-
Kan media to an estimated OD600 of 0.07. The culture was incubated at 37 °C, 170 r.p.m. until
2
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reaching an OD600 of 0.5 at which protein expression was induced by 0.35 mM IPTG and
incubating the culture at 30 °C, 170 r.p.m. for additionally 6 h. Cells were harvested by
centrifugation (4500 r.p.m., 4 °C) and P450-BM3/2M was purified by utilizing the N-terminal
6
2
hexa-histidine-tag as previously described . Cells pellets were resuspended in loading buffer (50
mM K HPO /KH PO , 800 mM NaCl, pH 7.5) supplemented with Roche cOmplete, Mini
2
4
2
4
Protease Inhibitor (3 ml/g pellet). Cells were lysed by sonication (QSonica sonificator, Q125, 5 x
[
50 s, amplitude: 50%, pulse: 1 s on, 2 s off]) and cell debris was discarded by centrifugation
18.000 rpm, 4 °C for 35 min). Purification of P450-BM3/2M was performed by immobilized
(
metal affinity chromatography (IMAC, Ni-NTA agarose beads, Qiagen). After loading the crude
lysate, the column was washed with loading buffer followed by washing buffer A (50 mM
K HPO /KH PO , 800 mM NaCl, pH 6.2) and washing buffer B (50 mM K HPO /KH PO ,
2
4
2
4
2
4
2
4
8
00 mM NaCl, 250 mM glycine, pH 7.5). The enzyme was subsequently eluted by elution buffer
(
50 mM K HPO /KH PO , 80 mM L-histidine, pH 7.5) and the purity of fractions were analyzed
2
4
2
4
by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE, 10% Mini
PROTEAN TGX-gels, BioRad) (Figure S 30). Elution fractions were pooled before concentrating
and exchanging buffer to reaction buffer (50 mM K HPO /KH PO , pH 7.5) using an Amicon®
2
4
2
4
Ultra 15 mL Centrifugal Filters 30 kDa cut-off. Concentration was measured by Bradford63
Expedeon BradfordUltra kit).
(
Biotransformation to convert α-pinene into trans-sobrerol. 0.5 mLscale reactions in reaction
buffer were carried out in 2 mLEppendorf tubes with (-)-α-pinene (2mM), NADPH (0.2mM),
G6P (glucose-6-phosphate, 100mM),G6PDH (glucose-6-phosphate dehydrogenase,
2
22U/mL), DMSO (2%v/v) and purified P450-BM3/2M (4.16μM), catalase (578 U/mL).
Reactions were incubated at 30 °C, 500 rpm for 22h before extracting in 2 x 0.5 mLethyl acetate
and separating organic-from aqueous phase by centrifugation (10,000 g, 10 min, r.t.). Organic
2
2

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phases were analyzed by GC-MS (Shimadzu, GCMS-QP2010 Ultra) performed on an Rxi®-5ms
column(30 m, 0.25 mm [inner diameter], 0.25 μm [film thickness], RESTEK). The temperature
program was set at 70 °C, before increasing to 300 °C with a rate of 20 °C/min and finally
increased to 350 °C with a rate of 5 °C/min before holding at 350 °C for 10 min.
Biotransformation to convert trans-sobrerol into sobreryl methacrylate (SobMA). Trans-
sobrerol (0.1 g, 0.58 mmol, 1eq) and PS Fluorescence (0.13 g) were mixed and heated to 80 °C
under exclusion of light. The reaction was initiated by addition of vinyl methacrylate (10-40 eq)
and proceeded for 72 h. For purification, the lipase and unreacted sobrerol were removed by
filtration through a thin layer of silica. Afterwards, the filtrate was stirred at 60 °C with 100 mg
of PS Fluorescence and 2 ml of water for 2 h. The reaction mixture was then filtered through
celite, diluted with EtOAc, washed with sodium bicarbonate and reduced to afford SobMA
1
(
0.105 g, 0.441 mmol, 76% yield). H-NMR and GC-FID samples were taken, using deuterated
chloroform (CDCl ) as solvent for the NMR, and ethyl acetate as solvent for the GC-FID.
3
Products were detected by GC-MS analysis as previously described (Biotransformation to
1
convert of α-pinene into prepare trans-sobrerol). H NMR (400 MHz, CDCl ): δ 6.09 (d, 1H, J =
3
1
1
.7 Hz), 5.72 (d, 1H, J = 4.7 Hz), 5.53 (d, 1H, J = 1.6 Hz), 5.30 (t, 1H, J = 2.1 Hz), 2.23-2.15 (m,
H), 2.05-2.00 (m, 1H), 1.94 (s, 3H), 1.88-1.69 (m, 2H), 1.68 (s, 3H), 1.47 (dt, 1H, J = 3.9, 13.0
1
3
Hz), 1.44 (s, 1H), 1.17 (2 s, 6H); C NMR (100 MHz, CDCl ): δ 167.2, 136.7, 131.0, 127.8,
3
1
25.1, 71.8, 71.0, 39.6, 30.1, 27.2, 26.8 (two signals), 20.6, 18.3
Chemical Synthesis of Sobreryl Methacrylate (SobMA). Methacryloyl chloride (3.05 mL, 31.5
mmol) was dissolved in DMF (30 mL) on an ice-bath and added dropwise under stirring to an
ice-cold solution of trans-sobrerol (4.83 g, 28.4 mmol), triethyl amine (5.90 mL, 42.6 mmol), and
DMAP (0.693 g, 5.67 mmol) in DMF (50 mL). The solution was stirred for 23 hours during
which it was allowed to reach room temperature. The solution wad diluted with 200 mL ethyl
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acetate and washed with 2 x 200 mL HCl (aq., 1M), 2 x 200 mL NaHCO (aq., 10 % w/w), and
3
2
00 mL de-ionized water. The organic phase was dried with MgSO and filtered. The solid was
4
rinsed with 200 mL ethyl acetate and the combined organic phases were reduced. Purification by
MPLC (100% dichloromethane → dichloromethane / ethyl acetate 4:1) afforded the SobMA
(4.41 g, 18.7 mmol, 66 % yield). Spectroscopical data were in agreement with those for the
previously synthesized SobMA
As an alternative synthesis of SobMA, methacryloyl chloride (0.63 mL, 6.51 mmol) was
dissolved in 2-MeTHF (5 mL) on an ice-bath and added dropwise under stirring to an ice-cold
solution of trans-sobrerol (1.00 g, 5.89 mmol), triethyl amine (1.25 mL, 9.02 mmol), and DMAP
(0.144 g, 1.18 mmol) in 2-MeTHF (30 mL). The solution was stirred for 19 hours during which it
was allowed to reach room temperature. Upon work-up and purification as described above,
SobMA (0.80 g, 3.36 mmol, 57 % yield) was afforded. Spectroscopical data were again in
agreement with those for the previously synthesized SobMA.
Free-Radical Polymerization of SobMA. A solution of SobMA (0.824 g, 3.46 mmol) stored in
dichloromethane / ethyl acetate was reduced under vacuum to roughly 80 % w/w SobMA. DMF
(
2.90 mL, 1.20 M) was added and the solution was reduced under vacuum overnight. The
solution was transferred to a round flask equipped with a magnetic stirrer containing ACVA
0.0042 mg, 0.015 mmol). The flask was sealed with a rubber septum. The solution was bubbled
(
with argon for 60 minutes and subjected to 4 vacuum /argon cycles. The flask was put in an oil
o
bath pre-heated to 70 C and stirred for five hours. The polymerization was quenched by
subjecting the solution to air and the solution was then diluted with 2 mL DMF. The polymer was
precipitated by dropwise addition of the solution to 400 mL diethyl ether under stirring and the
suspension was then stirred overnight. The polymer was recovered by filtration, rinsed with 200
mL diethyl ether and dried under vacuum overnight.
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RAFT Polymerizations of SobMA. Specific ratios between the reagents are described in Table
for each RAFT-derived polymer. A solution containing SobMA (typically 0.8-1.0 g) in
1
dichloromethane /diethyl ether was reduced under vacuum to around 80 % w/w SobMA. The
solution was diluted with DMF (1.20 M with regards to SobMA) and reduced under vacuum
overnight. CPAD was added and the solution transferred to a round flask with ACVA and a
magnetic stirrer. The flask was sealed with a rubber septum and the solution bubbled with argon
for 60 minutes. After being subjected to 4 vacuum / argon cycles, the flask was put on an oil bath
o
pre-heated to 70 C. After stirring for 8 hours, the polymerization was quenched exposure to air
and then diluted with 2 mL DMF. The polymers were precipitated by dropwise addition of the
solution to 400 mL diethyl ether under stirring. The solution was stirred overnight, filtered, and
the recovered polymers rinsed with 200 mL diethyl ether. The polymers were then dried under
vacuum overnight.
Co-polymers were synthesized in a similar fashion. For the statistical co-polymers, the co-
monomers were bubbled with argon for 30 minutes in a separate round flask and added via a
syringe under argon atmosphere to the SobMA-containing solution just before the vacuum /
argon cycles. Statistical co-polymers containing MMA were precipitated twice in diethyl ether.
Statistical co-polymers containing BMA were precipitated twice in heptane/diethyl ether 3:1. The
block co-polymers were prepared by bubbling the co-monomers with argon for 30 minutes. The
co-monomers were transferred via a syringe under argon atmosphere to the polymerization
solution containing SobMA after 6 hours of polymerization and the polymerization then
proceeded for another 18 hours. The block co-polymer containing MMA was obtained after
precipitation in heptane and then re-precipitation in diethyl ether from dichloromethane. The
block co-polymer containing BMA was precipitated twice in heptane/diethyl ether 3:1.
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ATRP Polymerizations of SobMA. Specific ratios between the reagents are described in Table
for each ATRP-derived polymer. Acetone was dried for 6 h on molecular sieves (3Å) and
1
distilled. A solution containing SobMA (typically 0.8-1.0 g) in dichloromethane /diethyl ether
was carefully reduced under vacuum and transferred in a 5mL round bottom flask. The flask,
equipped with a magnetic stirrer, was sealed with a septum and degassed. Dry acetone was added
(70 wt% with regards to acetone) and the solution was cooled to 0 °C. HMTETA and EBrP were
added and the solution and degassed by applying three vacuum-argon cycles. The flask was then
quickly opened to add Cu(I)Cl, whereafter it was sealed and subjected to three additional
vacuum−argon cycles. The flask was immersed in an oil bath preheated to 50 °C to start the
polymerization. After the reaction, the polymerization mixture was quenched exposure to air and
then diluted with 2 mL acetone. The polymers were obtained after two precipitations in water and
one precipitation in diethyl ether (250 mL each).
Enzymatic Polymerization. The specific ratios for homo- and copolymers are adopted from A.
Gross 55 and listed in Table 1. For the synthesis of PSobMA, the mixture of SobMA and ethyl
acetate was dilutet in THF and reduced. This was repeated until the EtOAc was completely
removed. The mixtuere SobMA (2.94 mmol) and THF was then added under Argon to small
roundbottom flask and diluted with THF and deionized water to reach a final concentration of 3M
(
in 25/75 THF/H O). Hydrogen peroxide (0.045 mmol), and 2,4-pentanedione (0.068 mmol)
2
were added and the reaction was initiated by the addition of 0.11 ml HRP (80 mg/mL, 8.4 mg of
enzyme). The reaction was stirred at r.t. until the reaction mixture was to viscous to be stirred.
Then, the reaction mixture was slightly diluted with THF and precipitated in a large excess of
diethyl ether (or chilled MeOH for BMA copolymers). The precipitate was filtered and dryd
under vacuum.
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Preparation of PSobMA-based ) Coatings. Coatings cross-linked via thiol-ene chemistry were
prepared by dissolving PSobMAR50 (0.100 g) and TMTP (0.046 mL, 0.140 mmol, thiol/ene =
1
:1) in THF (0.200 mL, containing 5 mg/mL Irgacure 651). The solution was applied to a glass
substrate using a 150 μm applicator. Upon drying in a fume hood for 10 minutes, the coating was
cured via UV-irradiation (10 x 5 s., total dose = 28.8 J/cm2).
Coatings cross-linked via transetherification were prepared by dissolving PSobMAR50 (0.100 g),
p-Toluenesulfonic acid (PTSA,1 μl) and HMMM (0.022 g) in THF (0.200 mL). The solution was
applied to a glass substrate using a 150 μm applicator and dried in a fume hood for 10 minutes.
o
The coating was cured in an oven at 140 C for 20 minutes.
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