Enzyme-responsive materials: Chirality to program polymer reactivity

Article abstract of DOI:10.1002/anie.200702438

(Figure Presented) A programmed response: Enzyme-responsive materials have been prepared from enzymatically synthesized, enantiomerically pure monomers. The extent of the material's response is encoded within its chiral makeup. This code can be effectively read out by ab enzymatic process, which leads to a change in the thermal properties of the material. The picture shows esterification of chiral alcohol groups on a polymer backbone (green) with vinyl acetate (red).

Full text of DOI:10.1002/anie.200702438

Communications
DOI: 10.1002/anie.200702438
Enzyme Catalysis
Enzyme-Responsive Materials: Chirality to Program Polymer
Reactivity**
Christopher J. Duxbury, Iris Hilker, Stefaan M. A. de Wildeman, and Andreas Heise*
Enzymatic catalysis has been shown to be a highly powerful
tool within chemistry.[1] The in vitro use of enzymes stems
from the study and observation of their role in nature, where
they catalyze a vast array of different chemical transforma-
tions, often with unprecedented levels of enantio- or regio-
selectivity. Enzymes have also been employed in the synthesis
and modification of polymers.[2] However, in most examples
enzymes merely replace a chemical catalyst.
Herein, we report for the first time the successful synthesis
of an ERM based on enantioselectivity. Furthermore, we
demonstrate that the system fulfills all of the above require-
ments and that the polymer can be encoded and read out.
The initial challenge was to address the first requirement
and design and synthesize a suitable monomer/polymer
system that would be able to contain the programming
through chirality. This problem is directly linked to the second
requirement of finding an enzyme that retains a high
selectivity towards the chiral polymer. A suitable substrate/
enzyme pairing was proposed based on previous work: 1-
phenylethanol and lipase B from Candida antarctica. Hult
et al. showed that this enzyme has a very high selectivity for
(R)-1-phenylethanol over the S form,[5] expressed in a reac-
tivity ratio of 1.3 ” 106:1 in the hydrolysis of the corresponding
acetates, a selectivity that has been utilized in the synthesis of
chiral polyester.[6] Here, we designed a monomer based on the
styrene analogue of 1-phenylethanol, which can be polymer-
ized by free-radical polymerization.[7]
The synthesis of this monomer was achieved by the ketone
p-vinylacetophenone, which can be chemically reduced to
obtain the corresponding racemic secondary alcohol. We
anticipated that alcohol dehydrogenases (ADHs) might be
promising catalysts for this reduction. It has been observed
that ADHs, which in nature selectively oxidize alcohols to
ketones, can catalyze the reverse reaction under appropriate
conditions. Depending on the source of the enzyme, both R
and S selectivity can be found with ADHs. Although their
selectivity in the reduction of different aromatic ketones has
been demonstrated,[8] they have, to our knowledge, never
been employed for the synthesis of a vinyl monomer.
We chose two commercially available enantiocomple-
mentary ADHs, that is, Lactobacillus brevis (R selective) and
Thermoanaerobacter sp. (S selective). Both enzymes depend
on nicotinamide adenine dinucleotide phosphate (NADPH)
as a cofactor, which serves as reduction equivalent (Figure 1).
However, both enzymes are also able to oxidize isopropanol
for cofactor regeneration, and therefore only catalytic
amounts of NADP+ are needed. The reaction equilibrium is
driven by using an excess of isopropanol in buffer (20% v/v).
Moreover, the high isopropanol content facilitates the
solubilization of hydrophobic substrates, such as p-vinylace-
tophenone. Gram-scale reductions of p-vinylacetophenone
were performed in good yield. The enantiopurity of the
resulting chiral alcohols was excellent (ee > 99% by chiral
GC, Figure 1) and the unwanted enantiomer was not detect-
able in either reaction.
One area in which the specific advantages of enzymes are
utilized is that of enzyme-responsive materials (ERMs).
These substances are seen as a new class of smart materials
in which a selective enzymatic action creates a macroscopic
change in material properties.[3] According to Ulijn, these
materials hold great promise in biomedical applications, as
enzyme responsiveness allows for a mutual communication
between the material and the biological environment, similar
to natural biological materials.[3] A number of approaches to
ERMs are described in the literature, most of which comprise
relatively complex enzyme-sensitive units, such as amino acid
sequences. For example, responsive materials have been
reported and discussed for drug-delivery applications.[4]
Chirality, on the other hand, has not yet been utilized in
ERMs. This is surprising, because chirality plays a dominant
role as a recognition feature in biological interactions. In fact,
many enzyme/substrate combinations exhibit exceptionally
high biological selectivity, such that an analogy with the
binary code of information technology can be drawn. We are
interested in exploiting this high selectivity for the design of
functional materials that respond to the interaction with an
enantioselective enzyme with a property change. Moreover,
we are aiming for polymers in which the extent of the
property change can be encoded or programmed into the
material by its chiral composition. A successful system must
address three fundamental requirements: 1) chiral mono-
mers, which can be synthesized in high purity; 2) a suitable
enzyme that retains a high stereoselectivity towards the chiral
polymer; and 3) an accurate encoding and enzymatic readout
of the polymer.
[*] Dr. C. J. Duxbury, Dr. I. Hilker, Dr. S. M. A. de Wildeman, Dr. A. Heise
DSM Research
P.O. Box 18, 6160 MD Geleen (The Netherlands)
Fax: (+31)46-476-0508
E-mail: andreas.heise@dsm.com
[**] This work was supported by the Marie Curie Action EIF program
“Enzymatic Approach to Chiral Multifunctional Materials for
Advanced Applications” (EnzyMat; C.J.D. contract no. MEIF-CT-
2006-039256).
Copolymers of styrene and p-vinylphenylethanol were
prepared from these monomers over the whole range of
compositions from 100% R to 100% S. These copolymer
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Chemie
Figure 1. Enzymatic synthesis of enantiomerically pure p-vinylphenyl-
ethanol and the chromatograms obtained from chiral GC of the
resulting S and R monomers (top and middle traces, respectively) and
the chemically synthesized racemic monomer (bottom trace).
Scheme 1. Copolymerization of the chiral monomers with styrene to
form the chiral copolymers used for subsequent R-selective enzymatic
grafting with vinyl acetate.
backbones were designed to contain a total of approximately
45% of the secondary alcohol monomer (either R, S, or a
mixture)—the programming that would subsequently deter-
mine the extent of the enzyme response. All polymers were
synthesized by free-radical polymerization to give products
with number-average molecular weight Mn = 5000–
6000 gmolÀ1 and polydispersity = 1.7–2.1. Differential scan-
ning calorimetry (DSC) analysis was carried out on all of the
polymer backbones. A similar glass transition temperature Tg
of approximately 1158C was found for all polymers, irrespec-
tive of their chiral composition, which shows that the
enantiomeric composition (that is, the programming) has no
effect on their thermal properties. Optical rotation measure-
ments of the polymers increased linearly from À20 to + 208,
which indicates that the R and S monomers had been
incorporated in the final polymers as expected.[7]
grafting was detected over a period of 24 h (Figure 2). Even
when the reaction was allowed to proceed for 48 h, there was
Once the chiral polymers had been prepared, the next step
was to determine whether this system fulfills the second
requirement and prove that the lipase retains a high
selectivity for the secondary alcohols, even when they are
distributed along a polymer chain. An immobilized form of
lipase B from C. antarctica (Novozym-435) was used to
catalyze the esterification of the alcohol groups on the
polymer backbone with vinyl acetate in toluene (Scheme 1).
The extent of the grafting from the secondary alcohol groups
Figure 2. Progression of the selective enzymatic grafting of vinyl
acetate for polymer backbones with a range of enantiomeric composi-
tions.
still no evidence of any grafting occurring from the backbone.
In contrast, when a backbone containing 100% R groups was
used, the enzymatic esterification of vinyl acetate occurred
from 75% of the alcohol groups within 24 h. This was
determined to be the maximum, as even after greatly
extended reaction times and the addition of fresh enzyme
and vinyl acetate, the conversion of secondary alcohol groups
could not be significantly increased, thus indicating that there
is some limiting factor in determining the maximum level of
grafting. This may be a result of some form of steric hindrance
1
was quantified by HNMR spectroscopy, where a clear shift
could be seen from d = 4.8 to 5.8 ppm on esterification of the
alcohol.[7]
When a backbone containing 100% S groups (styrene
copolymer containing approximately 45% alcohol monomer)
was used for the enzymatic grafting of vinyl acetate, no
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Communications
caused by the alcohol groups being located on a polymer
chain.[9]
Comparison of the grafting that occurs with the 100%
R backbone and the 100% S backbone shows that the
extremely high selectivity of the lipase towards the 1-phenyl-
ethanol moiety is retained, even in analogous polymer
reactions. This has never previously been shown. Moreover,
the binary reactivity of this system now allows for the design
of polymers with predetermined reactivity by copolymeriza-
tion of the two enantiomers.
information encoded in the polymer backbone, whilst the
DSC results demonstrate that this reading of the encoded
information also produces a change in the thermal properties
of the polymer.
We have demonstrated a novel, simple route to the
synthesis of enantiomerically pure monomers that can be
used to form polymers containing information encoded in
their chiral composition. By using the selectivity of an
enzyme, this information can be accurately read out, which
leads to a change in the thermal properties of the polymer.
To further investigate the programming that could be
encoded in the polymer, esterification reactions were carried
out on polymer backbones containing mixtures of R and
S alcohol groups. When the enzymatic esterification was
carried out on a backbone containing a racemic mixture, vinyl
acetate reacted with approximately 45% of the initiating
groups, in agreement with the previous levels of grafting.
Correspondingly, the use of a polymer backbone containing
25% R and 75% S groups led to reaction of less than 30% of
the secondary alcohol groups, whilst increasing the backbone
R content to 75% produced an appropriate increase in the
level of esterification to more than 55%.
DSC analysis of the polymers showed that the grafting of
vinyl acetate caused a decrease in the Tg of the polymers—the
higher the level of grafting, the larger the decrease in Tg
(Figure 3). The results of NMR and thermal analysis address
the third fundamental requirement described earlier. NMR
analysis shows that the enzyme can accurately read out the
Received: June 5, 2007
Published online: October 5, 2007
Keywords: chirality · enantioselectivity · enzyme catalysis ·
.
enzyme-responsive materials · polymerization
[1] K. Faber, Biotransformations in OrganicChemistry , Springer,
Berlin, 2000.
[2] a) S. Kobayashi, H. Uyama, S. Kimura, Chem. Rev. 2001, 101,
3793 – 3818; b) R. A. Gross, A. Kumar, B. Kalra, Chem. Rev. 2001,
101, 2097 – 2124; c) I. K. Varma, A. C. Albertsson, R. Rajkhowa,
R. K. Srivastava, Prog. Polym. Sci. 2005, 30, 949 – 981.
[3] R. V. Ulijn, J. Mater. Chem. 2006, 16, 2217 – 2225.
[4] a) Z. Yang, H. Gu, D. Fu, P. Gao, J. K. Lam, B. Xu, Adv. Mater.
2004, 16, 1440 – 1444; b) P. D. Thornton, R. J. Mart, R. V. Ulijn,
Adv. Mater. 2007, 19, 1252 – 1256.
[5] A. O. Magnusson, M. Takwa, A. Hamburg, K. Hult, Angew.
Chem. 2005, 117, 4658 – 4661; Angew. Chem. Int. Ed. 2005, 44,
4582 – 4585.
[6] a) I. Hilker, G. Rabani, G. K. M. Verzijl, A. R. A. Palmans, A.
Heise, Angew. Chem. 2006, 118, 2184 – 2186; Angew. Chem. Int.
Ed. 2006, 45, 2130 – 2132; b) J. van Buijtenen, B. A. C. van As, J.
Meuldijk, A. R. A. Palmans, J. A. J. M. Vekemans, L. A. Hulshof,
E. W. Meijer, Chem. Commun. 2006, 3169 – 3171; c) B. A. C.
van As, J. van Buijtenen, A. Heise, Q. B. Broxterman, G. K. M.
Verzijl, A. R. A. Palmans, E. W. Meijer, J. Am. Chem. Soc. 2005,
127, 9964 – 9965.
[7] Supporting Information available: experimental procedures,
NMR spectrum, and optical rotation data.
[8] a) W. Hummel, Adv. Biochem. Eng. Biotechnol. 1997, 58, 145 –
184; b) K. Nakamura, R. Yamanaka, T. Matsuda, T. Harada,
Tetrahedron: Asymmetry 2003, 14, 2659 – 2681; c) W. Kroutil, H.
Mang, K. Edegger, K. Faber, Curr. Opin. Chem. Biol. 2004, 8,
120 – 126.
[9] a) C. J. Duxbury, D. Cummins, A. Heise, Macromol. Rapid
Commun. 2007, 28, 235 – 240; b) M. Hans, P. Gasteier, H. Keul,
M. Moeller, Macromolecules 2006, 39, 3184 – 3193.
Figure 3. DSC Tg analysis of the chiral polymer backbone before (gray)
and after (white) selective enzymatic grafting of vinyl acetate (for 24 h)
as a function of chiral composition.
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