A Breathing Atom-Transfer Radical Polymerization: Fully Oxygen-Tolerant Polymerization Inspired by Aerobic Respiration of Cells

Article abstract of DOI:10.1002/anie.201711105

The first well-controlled aqueous atom-transfer radical polymerization (ATRP) conducted in the open air is reported. This air-tolerant ATRP was enabled by the continuous conversion of oxygen to carbon dioxide catalyzed by glucose oxidase (GOx), in the presence of glucose and sodium pyruvate as sequential sacrificial substrates. Controlled polymerization using initiators for continuous activator regeneration (ICAR) ATRP of oligo(ethylene oxide) methyl ether methacrylate (OEOMA, M_n=500) yielded polymers with low dispersity (1.09≤?≤1.29) and molecular weights (MWs) close to theoretical values in the presence of pyruvate. Without added pyruvates, lower MWs were observed due to generation of new chains by H₂O₂ formed by reaction of O₂ with GOx. Successful chain extension of POEOMA₅₀₀ macroinitiator with OEOMA₃₀₀ (?≤1.3) and Bovine Serum Albumin bioconjugates (?≤1.22) confirmed a well-controlled polymerization. The reactions in the open air in larger scale (25 mL) were also successful.

Full text of DOI:10.1002/anie.201711105

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Accepted Article
Title: A Breathing ATRP: Fully Oxygen Tolerant Polymerization
Inspired by Aerobic Respiration of Cells
Authors: Krzysztof Matyjaszewski, Liye Fu Fu, Alan Russell Russell,
and Alan E. E. Enciso
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201711105
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10.1002/anie.201711105
Angewandte Chemie International Edition
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A Breathing ATRP: Fully Oxygen Tolerant Polymerization Inspired
by Aerobic Respiration of Cells
Alan E. Enciso†, Liye Fu†, Alan J. Russell and Krzysztof Matyjaszewski*
Abstract: The first well-controlled aqueous atom transfer radical
polymerization (ATRP) conducted in the open air is reported. This air
tolerant ATRP was enabled by the continuous conversion of oxygen
to carbon dioxide catalyzed by glucose oxidase (GOx), in the
presence of glucose and sodium pyruvate as sequential sacrificial
substrates. Controlled polymerization using initiators for continuous
activator regeneration (ICAR) ATRP of oligo(ethylene oxide) methyl
ether methacrylate (OEOMA, Mn=500), yielded polymers with low
dispersity (1.09≤Đ≤1.29) and molecular weights (MW) close to
theoretical values in the presence of pyruvate. Without added
pyruvates, lower MW were observed due to generation of new chains
by H2O2 formed by reaction of O2 with GOx. Successful chain
extension of POEOMA500 macroinitiator with OEOMA300 confirmed a
well-controlled polymerization (Đ≤1.3). The reactions in open air in
larger scale (25 mL) were also successful. In addition, a “grafting from”
polymerization was carried out from ATRP initiators covalently
attached to Bovine Serum Albumin (BSA) with low amounts of catalyst
(ppm vs. monomer) and short reaction time (≤ 2 hours).
feasibility of polymerizations in open air in the presence of the
enzyme, which has also been successfully carried out by other
groups.[20, 21] However, the generated hydrogen peroxide (from
glucose oxidase-catalysed step between oxygen and D-glucose),
[22-24] was not removed and its fate has not been addressed.[25]
In an ATRP, hydrogen peroxide can oxidize Cu(I) activators
and also it can initiate new chains via a Fenton-like reaction.
Therefore, the enzyme-assisted deoxygenating strategy in ATRP
is more challenging, as it should also include elimination of H2O2.
Initially, we studied the GOx-catalyzed oxygen removal in an
Initiators for Continuous Activator Regeneration Atom Transfer
Radical Polymerization (ICAR ATRP) in aqueous systems[26]
using 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride
(VA-044), glucose (200 mM), GOx (2 μM)[17] and NaBr (100 mM)
in open air. Interestingly, the first attempts performed with stirring
in completely opened vials did not yield any polymerization.
However, in the absence of stirring, successful polymerizations
were achieved. This indicated the importance of oxygen diffusion
in the reaction, as previously reported.[18] Therefore, reactions in
capped vials were carried out in order to avoid constant oxygen
diffusion into the system, providing reproducible polymerizations,
even with rapid stirring.
The initial polymerizations were conducted with a molar ratio
of [OEOMA500]/[Initiator]/[VA-044]/[Cu]/[TPMA]=200:1:0.3:0.2:0.4,
using 20 vol.% monomer in 1X phosphate buffer saline (PBS) at
45°C (Figure S1). 2-Hydroxyethyl-bromoisobutyrate (HO-EBiB)
was the initiator, yielding polymers with relatively low dispersity,
(Ð~1.27). However, the number average molecular weights (Mn)
were approximately 4 times lower than the theoretical values. This
indicated formation of new chains, plausibly generated by H2O2.
The use of α-Bromophenylacetic acid (BPAA), a more active
initiator,[27] did not significantly improve control.
ATRP is one of the most widely used Reversible
Deactivation Radical Polymerization (RDRP) techniques due to
the large range of available monomers, initiators and catalysts
that allow the synthesis of polymers with complex architectures
and multiple functionalities.[1-4] The synthesis of polymers under
aqueous conditions via ATRP was employed to grow polymers
from biomacromolecules,[5-9] Although such polymerizations have
been successful, one particularly vexing challenge is that ATRP is
sensitive to oxygen and cannot be performed in open vessels. In
the laboratory, ATRP is generally performed in a deoxygenated-
capped container, or in the presence of excess reducing agents
to remove limited amounts of air present in closed flasks.[10-12]
Oxygen is a diatomic molecule with two unpaired electrons
in its outer shell that quickly capture radicals, forming relatively
stable peroxy radicals, therefore stopping the polymerizations.[13,
Additionally, different ratios of reactants were explored to
enhance polymerization control. Experiments with different
loadings of catalyst (100, 300, 500 and 1000 ppm) revealed that
the amount of Cu can be reduced to 300 ppm; but at lower
concentrations, the dispersity increased (Figure S2). Reducing
the ratio of VA-044 to alkyl halide initiator (0.3, 0.1, 0.05, 0.02, and
0.01) slowed down the reaction and diminished control below the
ratio 0.05 (Figure S3). Since the reactions were performed in 1X
PBS, which contains 137 mM NaCl, no additional NaBr salt was
added, as reported for other aqueous ATRP systems.[9] The
addition of salt at concentrations higher than 100 mM slowed
down the reaction (Figure S4).
14]
Thus, oxygen is the undesired radical scavenger in radical
polymerizations. To overcome this, deoxygenation with inert
gasses, use of a glove box or other techniques such as “freeze,
pump, thaw” are used for the effective removal of oxygen.[15]
Previously, Yagci[16] and Stevens[17-19] described the use of
glucose oxidase (GOx) as an effective scavenger for oxygen in
free radical polymerization and in the denoted enzyme-assisted
reversible
addition-fragmentation
chain-transfer
(RAFT)
polymerizations, respectively. They also demonstrated the
[*] Dr. A. E. Enciso,[+] L. Fu,[+] Prof. K. Matyjaszewski
Department of Chemistry, Carnegie Mellon University
4400 Fifth Avenue, Pittsburgh, PA 15213 (USA)
E-mail: km3b@andrew.cmu
When higher molecular weights were targeted, i.e degrees
of polymerization (DPn) >200, the obtained experimental MW, Mn
= 68,000; which was nearly 5 times lower than Mn,Th = 300,000,
although all monomer was completely consumed (Table 1,
entries 1-5). This suggested the generation of new chains from
H2O2 produced by GOx, continued even in a closed vial. The
formed H2O2 could further have reacted with CuI species via a
Fenton-like reaction, yielding initiating radicals and new polymer
chains[28-31] (Scheme S2). To prevent formation of new chains, the
reactive oxygen species should be eliminated and converted to
Prof. A. J. Russell
Department of Chemical Engineering, Carnegie Mellon University
5000 Forbes Avenue, Pittsburgh, PA 15213 (USA)
E-mail: alanrussell@cmu.edu
[†] These authors contributed equally to this work
Supporting information for this article is given via a link at the end of the
document.
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10.1002/anie.201711105
Angewandte Chemie International Edition
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inert products that do not interfere with propagating radicals or
catalyst.[32]
Table 1. ICAR ATRP targeting various DPn with 300 ppm Cu with (+) and without
(-) 100 mM sodium pyruvate
M/I/VA-044/
Cu/TPMA
Sodium
pyruvate Time (h) (%)
RXN
Conv
In some biological systems, such as in the aerobic
respiration of cells, glucose and oxygen after several enzyme-
catalyzed reactions produce CO2 and ATP molecules. At the first
stage, glucose proceeds through a series of transformations in
the glycolysis cycle, which yields pyruvate and ATP, then pyruvate
reacts with oxygen to generate acetyl-CoA that in the Krebs cycle
forms CO2 and additional ATP molecules (Scheme 1A).
Entry
Mn,Th×10-3 Mn,exp×10-3
Ð
1
2
100/1/0.3/0.03/0.15
200/1/0.3/0.06/0.3
400/1/0.3/0.12/0.6
600/1/0.3/0.18/0.9
800/1/0.3/0.24/1.2
100/1/0.3/0.03/0.15
200/1/0.3/0.06/0.3
400/1/0.3/0.12/0.6
600/1/0.3/0.18/0.9
800/1/0.3/0.24/1.2
2
3
51
95
90
50
76
92
95
97
94
96
25.5
95.3
180
152
304
45.8
95.4
194
283
384
23.5
96.8
110
1.25
1.27
1.23
1.33
1.24
1.15
1.17
1.17
1.18
1.27
-
-
3
2
-
4
2
73.3
67.7
82.7
111
-
5
2
-
6
1.5
2
+
+
+
+
+
7
8
1.5
1.5
1.5
239
336
442
9
10
M = OEOMA500, [M] = 10 vol % in 1X PBS, [NaBr] = 100 mM, [Glucose] = 200
mM, [GOx] = 2 μM, [Sodium Pyruvate] = 100 mM, T = 45oC.
This experiment confirmed the retention of chain end-functionality
in the “breathing” ICAR ATRP.
Thermoregulation in this system (Figure 2C) was confirmed
by temporal on/off experiments consisting on heating at 45 °C and
cooling the system in iced water in 15 minutes intervals for 2 hours.
VA-044 at ~44 °C has appropriate decomposition rate (t1/2 = 10 h).
Therefore, cooling the reaction in ice bath (0°C) essentially
stopped the decomposition of VA-044 and generation of radicals,
quenching polymerization and demonstrating possibility of
temporal control. The final polymer displayed Mn matching the
theoretical value and low dispersity (Figure 2D). Reactions were
also carried out in open air in a 50 mL round bottom flask. They
yielded polymers with dispersities and molecular weights identical
to the model reactions (Figure S5 and S6), indicating good
scalability.
To test the possibility of using this bio-inspired cascade
system to modify biomolecules, the synthesis of protein-polymer
hybrids was carried out by grafting from the surface of Bovine
Serum Albumin (BSA). An ATRP initiator containing a cleavable
ester moiety and anchoring N-hydroxysuccinimide (NHS) species
was synthesized and covalently coupled to the accessible lysine
residues on the surface of BSA in PBS.[9] The polymerization was
Scheme 1. A) Aerobic respiration in cells. B) Breathing ATRP.
We decided to develop a bio-inspired polymerization system
(Scheme 1B), in which GOx first catalyzed the conversion of
glucose and oxygen into D-glucono-1,5-lactone and hydrogen
peroxide and therefore eliminated oxygen inhibition of a radical
polymerization. Then, in the second step, we reacted the toxic
byproduct of oxygen removal, hydrogen peroxide, with sodium
pyruvate (k = 3.5 × 105 M−1 sec−1)[33] yielding CO2, acetate and
water.[34] This green approach permits ATRP in open air by
continuous elimination of oxygen without generation of new
chains. The products did not interfere with propagating radicals or
catalysts and do not generate new chains.
Experiments using the improved bio-deoxygenation
conditions (Table 1, entries 6-10) for different targeted DPn (100,
200, 400, 600 and 800) formed polymers with low dispersity (1.15
≤ Đ ≤ 1.27) and reached quantitative conversions in less than 2
hours.
Chain extension was accomplished by either the direct
addition of OEOMA300 monomer (Figure 2A) to the vial after the
first block reached ~95% conversion (monitored by NMR) or by
stopping the reaction at 60% conversion, followed by dialysis in
deionized water with a 5kD MW Cut-off membrane and
subsequent lyophilization. The dry macroinitiator was redissolved
to continue the polymerization of the second block (Figure 2B).
p(OEOMA500
)
p(OEOMA500)
A)
B)
Mn = 64,100, Mw/Mn = 1.18
p(OEOMA500)-b-p(OEOMA300
Mn = 93,600, Mw/Mn = 1.30
Mn= 42,300, Mw/Mn=1.23
p(OEOMA500)-b-p(OEOMA300
Mn= 79,400, Mw/Mn=1.28
)
)
104
105
Molecular Weight
106
104
105
106
Molecular Weight
D)
Mn = 107,200
Mn,Th = 89,300
Mw/Mn = 1.16
104
105
Molecular Weight
Figure 2: Chain extension experiments via A) One-pot strategy w/ pyruvate in
open air; B) Purification-reinitiation strategy. C) Kinetic plots of temporal control
of on-off study under different temperatures (45oC vs. ice bath) and D) GPC
trace of final polymer product.
Figure 1. Corrected Mn (solid points), Mn,Th (dashed lines) and dispersities
(hollowed points) for Table 1 (Left: entries 1-5; Right: entries 6-10).
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Keywords: Oxygen • ATRP • Glucose Oxidase • Hydrogen
Peroxide • Pyruvate
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proceeded under “breathing” ICAR ATRP conditions (Figure 3).
After the polymerization of OEOMA500 monomer was completed,
the polymer was cleaved from the enzymatic structure under mild
basic conditions (5% NaOH) and analyzed via THF GPC,
confirming synthesis of well-defined polymers with predetermined
molecular weight (Mn= 81,300) and low dispersity (Ð = 1.22).
Model experiments were conducted to evaluate a reaction
between H2O2 and Cu(I) species. In a sealed and degassed
vessel, 1.25 μmol H2O2 was dissolved in 5 mL 1X PBS with or
without sodium pyruvate (100 mM) and then 2 mM of CuBr/TPMA
was added. Without pyruvate, a significant increase of absorption
at 870 nm was observed by UV-vis spectroscopy, indicating
formation of Cu(II)/TPMA complex (ε[870] = 222 M-1cm-1 in water,
Figure S7). In the experiment with pyruvate, the Cu(II)/TPMA
complex was not formed (Figure S8). Also, to mimic the
polymerization conditions, the same concentrations of glucose
and GOx with or without sodium pyruvate were dissolved in 3 mL
of 1X PBS in a cuvette without degassing; then Cu(I)/TPMA stock
solution was added. The results showed that the pyruvate
successfully suppressed the oxidation of Cu(I), even in a flask
open to air (Figure S9).
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Acknowledgements
Financial support from NSF (CHE 1707490) is acknowledged.
Mateusz Olszewski‘s help with ToC graphic design is appreciated.
A.E.E. acknowledges the Mexican Council for Science and
Technology - CONACyT for the posdoctoral award (291231 - 2do
año de Continuidad de Estancias Posdoctorales en el Extranjero
Vinculadas a la Consolidación de Grupos de Investigación)
CMU provided support to the CMU Center for Polymer Based
Protein Engineering.
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
Alan E. Enciso†, Liye Fu†, Alan J. Russell
and Krzysztof Matyjaszewski*
Well-controlled
aqueous
ATRP
conducted in the open air was enabled by
the continuous conversion of oxygen to
carbon dioxide catalyzed by glucose
oxidase (GOx), in the presence of
glucose and sodium pyruvate as
sequential sacrificial substrates. Without
pyruvate lower molecular weights were
observed due to generation of new
chains by H2O2 formed by reaction of O2
with GOx. Block copolymerizations and
synthesis of protein polymer hybrids were
achieved with ppm amounts of Cu
catalyst.
Page No. – Page No.
Title: A Breathing ATRP: Fully Oxygen
Tolerant Polymerization Inspired by
Aerobic Respiration of Cells
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