Enzymatic Cascade Catalysis for the Synthesis of Multiblock and Ultrahigh-Molecular-Weight Polymers with Oxygen Tolerance

Article abstract of DOI:10.1002/anie.201707993

Synthesis of well-defined multiblock and ultrahigh-molecular-weight (UHMW) polymers has been a perceived challenge for reversible-deactivation radical polymerization (RDRP). An even more formidable task is to synthesize these extreme polymers in the presence of oxygen. A novel methodology involving enzymatic cascade catalysis is developed for the unprecedented synthesis of multiblock polymers in open vessels with direct exposure to air and UHMW polymers in closed vessels without prior degassing. The success of this methodology relies on the extraordinary deoxygenation capability of pyranose oxidase (P2Ox) and the mild yet efficient radical generation by horseradish peroxidase (HRP). The facile and green synthesis of multiblock and UHMW polymers using biorenewable enzymes under environmentally benign and scalable conditions provides a new pathway for developing advanced polymer materials.

Full text of DOI:10.1002/anie.201707993

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Accepted Article
Title: Enzymatic Cascade Catalysis for the Synthesis of Multiblock and
Ultrahigh-Molecular-Weight Polymers with Oxygen Tolerance
Authors: Zesheng An, Zhifen Liu, and Yue Lv
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201707993
Angew. Chem. 10.1002/ange.201707993
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10.1002/anie.201707993
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Enzymatic Cascade Catalysis for the Synthesis of Multiblock and
Ultrahigh-Molecular-Weight Polymers with Oxygen Tolerance
Zhifen Liu,† Yue Lv,† and Zesheng An*
Abstract: Synthesis of well-defined multiblock and ultrahigh-
molecular-weight (UHMW) polymers has been a perceived challenge
for reversible-deactivation radical polymerization (RDRP). An even
more formidable task is to synthesize these extreme polymers in the
presence of oxygen. A novel methodology involving enzymatic
cascade catalysis is developed for the unprecedented synthesis of
multiblock polymers in open vessels with direct exposure to air and
UHMW polymers in closed vessels without prior degassing. The
success of this methodology relies on the extraordinary
deoxygenation capability of pyranose oxidase (P2Ox) and the mild
yet efficient radical generation by horseradish peroxidase (HRP).
The facile and green synthesis of multiblock and UHMW polymers
using biorenewable enzymes under environmentally benign and
deoxygenation,[1b, 4e, 4g, 7-8] there has been no literature reports on
the controlled synthesis of multiblock polymers in open vessels
with direct exposure to air and UHMW polymers in closed
vessels without prior degassing.
We report a novel enzymatic catalysis cascade involving
P2Ox deoxygenation and HRP initiation for the unprecedented
synthesis of well-defined multiblock polymers in open vessels
and UHMW polymers in closed vessels both with oxygen
tolerance under mild conditions. Given the demand for green
and sustainable chemical processes and the difficulties in
synthesizing such extreme polymers, this biocatalysis strategy
represents a significant advance in polymer synthesis via RDRP,
with merits of mildness, high efficiency, environmental
friendliness, and possibility for scale-up.
scalable conditions provides
advanced polymer materials.
a new pathway for developing
Enzymatic catalysis has long been used in green synthesis
of polymers, but it is only recently that enzymes are engaged in
RDRP for synthesis of precision polymers.[9] P2Ox,
a
RDRP has revolutionized the field of polymer synthesis, allowing
general access to various polymer architectures with
predetermined molecular weights (MWs) and controlled
molecular weight distributions (MWDs).[1] Synthesis of multiblock
(≥ 5 blocks) and UHMW (~ 106 g/mol) polymers with low MWDs
has been considered a challenge due to increased termination
during extended polymerization times or on multiple initiations.[2]
Significant advances have recently been made to address these
synthetic limitations via development of new methodologies and
manipulation of the kinetics of existing techniques. Multiblock
polymers with a precise sequence order are promising for use in
biorecognition, catalysis, ordered materials and information
storage.[3] Enhanced livingness of RDRP has enabled the
preparation of well-defined multiblock polymers, which has been
achieved primarily by means that lowers the concentration of
homotetrameric flavoprotein widely distributed in root fungi,
reduces oxygen using a number of saccharides to generate
H2O2, which is then supplied to peroxidases as an oxidant to
decompose lignin.[10] Inspired by this enzymatic cascade in
nature, we designed a novel P2OxHRP catalysis cascade for
controlled reversible additionfragmentation chain transfer
(RAFT) polymerization with oxygen tolerance (Scheme 1).
Specifically, P2Ox reduces oxygen with D-glucose to generate
H2O2, which is subsequently used by HRP to produce
acetylacetone
(ACAC)
radicals
to
initiate
RAFT
polymerization.[11] This work is the first use of P2Ox for
deoxygenation in a RDRP. In comparison with the more
commonly used GOx,[8b, 8e] P2Ox has several key advantages
including a broader range of saccharide substrates, no selection
of anomers, a remarkably higher affinity for both glucose and
oxygen, and importantly, a much higher enzyme efficiency
(Table S1).[12] Furthermore, P2Ox acts on the carbon-2 position
of D-glucose, producing 2-dehydro-D-glucose, which is fairly
stable against hydrolysis, while D-glucono--lactone generated in
GOx deoxygenation is readily hydrolyzed into gluconic acid, thus
gradually reducing pH of the solution (Figure S1). We
conducted polymerization in buffered aqueous solution (pH ~ 7)
at 30 °C with the ratios of reagents being carefully optimized to
allow excellent RAFT control to be achieved. Under these
conditions, both P2Ox and HRP showed relatively high retention
of activities (Figure S2). In a typical polymerization, a solution
containing monomer, chain transfer agent (CTA), glucose, and
ACAC was deoxygenated by P2Ox for 10 min to generate
sufficient H2O2 (~ 2.68 mM) (Figure S3), followed by addition of
HRP to initiate polymerization in either open or closed vessels.
active radicals while maintaining
a
reasonably high
polymerization rate.[4] Well-defined UHMW polymers hold great
potential as advanced materials such as high-performance
hydrogels and photonic crystals.[5] Maximizing the kinetic chain
length has been realized by devising polymerization systems
that favor a high kp/kt ratio.[6]
RDRP techniques are well-known to be highly sensitive to
the presence of oxygen. Well-defined multiblock and UHMW
polymers have predominantly been prepared under stringent
oxygen-free conditions.[7] Conducting RDRP in the presence of
oxygen for the synthesis of such difficult polymers certainly adds
another level of challenge to conquer. Although several
strategies have been employed to overcome this oxygen-
constraint to certain degrees, including the use of sacrificial
reductants, photocatalysts, and glucose oxidase (GOx)
Z. Liu, Y. Lv, and Prof. Z. An
Institute of Nanochemistry and Nanobiology
College of Environmental and Chemical Engineering
Shanghai University, Shanghai 200444, China
E-mail: an.zesheng@shu.edu.cn
†These authors made equal contribution
Supporting information for this article is given via a link at the end of
the document.
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Scheme 1. Schematic Representation of P2OxHRP Cascade Catalysis for RAFT Polymerization with Oxygen Tolerance and Chemical Structures of Chain
Transfer Agent (CTA) and Monomers (M).
in an open vessel using the P2OxHRP cascade catalysis had
all the characteristics of a well-controlled RAFT process.
Figure 1. Polymerization kinetics of DMA conducted in an open vessel:
[CTA]:[DMA]:[HRP]:[ACAC]=1:200:0.00054:1, [glucose]=80 mM, [P2Ox]=2.7
U/mL, [DMA]=10% w/v, PBS (pH 7) 5 mL, 30 °C , stirring speed 100 rpm,
prepolymerization incubation time 10 min. (A) Pseudo-first-order kinetic plot of
ln([M]0/[M]) vs time. (B) Evolution of MW and dispersity with conversion (RI-
GPC, DMF, PMMA standards).
The feasibility of conducting well-controlled RAFT
polymerization in open vessels using this P2OxHRP cascade
catalysis was first examined with N,N-dimethylacrylamide (DMA).
As shown in Figure 1, polymerization kinetic study indicated that
after an initial induction period of approximately 10 min the
polymerization showed an excellent linearity for ln([M]0/[M]) vs
time and hence a pseudo-first-order polymerization kinetics. The
initial induction period was attributable to further deoxygenation
of the solution by P2Ox before the polymerization proceeded
Figure 2. Results of polymerizations of DMA, PEGA, and PEGMA in PBS (pH
7) targeting different DPs conducted in open vessels. (A) Dependence of MW
and dispersity on DP. (B-D) GPC traces of PDMAs, PPEGAs, and PPEGMAs
(RI-GPC, DMF, PMMA standards).
under
a
stable radical flux condition. Gel permeation
chromatography (GPC) measurements showed that the polymer
samples carefully withdrawn at different times during the
polymerization had symmetric and monomodal traces with no
obvious tailing being observed (Figure S6), suggesting that
oxygen removal was indeed effective and premature termination
by oxygen was negligible. MWDs gradually narrowed as the
polymerization proceeded and low dispersities (Đ <1.2) were
obtained at high conversions, indicating that the polymerization
Next we conducted independent polymerizations targeting
different degrees of polymerization (DP) for three types of water-
soluble monomers, including DMA, poly(ethylene glycol) methyl
ether acrylate (PEGA), and poly(ethylene glycol) methyl ether
methacrylate (PEGMA) in open vessels; the results are shown in
Figure 2. In most cases, high monomer conversions (> 90%)
were achieved in 2 h. For PDMAs with target DPs up to 1000,
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10.1002/anie.201707993
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low dispersities (Đ < 1.2) were achieved in all cases. PPEGA
and PPEGMA had higher dispersities with Đ ~1.3 when targeting
a DP of 200, their dispersities further increased to 1.45 and 1.49,
respectively, when targeting a high DP of 500. The higher
dispersities of PPEGAs and PPEGMAs when compared to
PDMAs were understandable considering that their monomer
units bear oligomeric ethylene glycol side chains, which present
large steric hindrance for polymerization. These PPEGA and
PPEGMA polymers already had a measured MW of 133 and
166 kg/mol, respectively, which are relatively high MWs among
polymers commonly synthesized by RAFT.
Figure 3. 1H NMR spectra in D2O, GPC traces (RI-GPC, DMF, PMMA standards), MW and dispersity of each blocking cycle for the synthesis in PBS (pH 7) of
PDMA150-(PAML50-PDMA50)4-PAML50 (A) and PDMA100-(PAML50-PAML50-PDMA50)3 (B) decablock copolymers in open vessels.
with a first block having a DP of 100 or 150 and then a DP of 50
for each subsequent blocking. It is worth emphasizing that for
multiblock polymers as these reported herein, these DPs are
relatively high. Four multiblock (co)polymers have been
prepared using this technique, including PDMA150-(PDMA50)9
and PAML150-(PAML50)9 quasi decablock homopolymers,
PDMA150-(PAML50-PDMA50)4-PAML50 and PDMA100-(PAML50-
PAML50-PDMA50)3 decablock copolymers (Table 1). The
efficiency of the multiblocking process in open vessels was first
tested with PDMA and PAML homopolymers (Figure S10). Each
block was allowed to achieve a very high monomer conversion
as indicated by 1H NMR spectroscopy analysis. As the block
number increased, the GPC traces clearly shifted to higher MWs,
each showing nicely symmetric and monomodal MWDs. The
dispersity was observed to gradually increase as the block
Having demonstrated the robust nature of the P2OxHRP
cascade catalysis for efficient synthesis of well-defined
homopolymers with oxygen tolerance, we next tackled the
challenge of synthesizing multiblock (co)polymers in open
vessels. These multiblock (co)polymers were prepared via an
iterative strategy by sequential addition of monomers. Note that
it was necessary to supplement enzymes, glucose and ACAC
along with the addition of new monomers for each blocking cycle
to maintain the ability for continuous deoxygenation and radical
generation, and the reaction vessel remained open to air
throughout the whole synthetic process (Tables S8-11). Two
acrylamides, DMA and 4-acryloylmorpholine (AML), were used
to demonstrate the synthesis of multiblock polymers for their
high kp and low propensity to chain transfer. We typically started
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