An Honors Program Thesis Proposal
Synthesis of Low Molecular Weight and Low Polydispersity
Poly(Methacrylic Acid) for Studies of the pH-Dependent
Conformational Transition in Aqueous Solution
Author: Danielle Petko
Advisor: Dr. Devon Shipp
April 19, 2005
Objective
To synthesize several monodisperse samples of poly(methacrylic acid) with molecular weights in
the range of 2,000 to 15,000, and to study the effects of molecular weight and polydispersity on
the pH-dependent conformational transition of the polymer in aqueous solution.
Purpose
Poly(carboxylic acid)s such as poly(acrylic acid) (PAA), poly(methacrylic acid) (PMA), and
poly(2-ethacrylic acid) (PEAA) are very widely used in industry as thickeners and flocculants.
However, poly(carboxylic acids) also have uses in the biological arena due to their pHresponsiveness. These compounds undergo a rod-to-globule conformational transition in
response to acidification [1]. The polymer’s globular form can be bound to vesicular
phosphatidylcholine membranes, changing the permeability of the membrane through membrane
reorganization. The membrane is then responsive to environmental cues such as pH,
temperature, glucose concentration and light [2]. This property gives way to possible uses in
gene therapy, targeted drug delivery, and signal amplification in biochemical assays [3].
In order for intracellular destinations to be targeted, the “critical pH” must be well defined [4].
This value can be controlled by either altering properties of the bilayer membrane or by varying
the polymer chain structure [4]. Tirrell’s group has done extensive work on the interaction of the
hydrophobic polyelectrolyte, poly(2-ethacrylic acid) (PEAA) with phospholipids bilayer
membranes. When PEAA is used with low molecular weights and low polydispersities there is
better definition of the “critical pH” than with higher molecular weights and polydispersities. It
has been found that the conformational transition of PEAA shifts to lower pH as the chain length
is reduced, and thus the molecular weight can be used to tune the “critical pH.” In order to
receive sharper conformational transitions, samples that are less polydisperse can be used [4].
A poly(carboxylic acid) that is very structurally similar to PEAA is PMA (Figure 1). These
compounds are both hydrophobic polyelectrolytes, and therefore have very similar properties.
However, PEAA can be much more difficult to synthesize and work with than PMA. At this
point, research on the pH-induced conformational changes of PMA has not been conducted using
low molecular weight polymers with low polydispersities. Studies have only been done for
polydisperse high molecular weight polymers [5]. It is hypothesized that PMA with low
molecular weights and polydispersities will have better definition of the “critical” pH.
HO
O HO
O HO
HO
O
O HO
O HO
O
Poly(methacrylic acid)
Poly(2-ethacrylic acid)
Figure 1. Structures of poly(2-ethacrylic acid) and poly(methacrylic acid)
2
Background
There are a number of polymerization techniques available for the synthesis of poly(methacrylic
acid), the most obvious of which is direct radical polymerization of methacrylic acid. However,
this technique provides difficulty in controlling the molecular weight and polydispersity of the
synthesized polymers. This is a problem because poly(methacrylic acid) samples with welldefined molecular weights and polydispersities are needed. To avoid the problems introduced by
direct polymerization, the synthesis can be performed by polymerizing “protected monomers”
and removing the protecting group in subsequent reaction steps. The living radical
polymerization (LRP) technique, reversible addition-fragmentation chain transfer (RAFT)
polymerization, will be used to polymerize the protected monomers, as this technique gives
monodisperse polymers of predictable molecular weight.
RAFT polymerization works under the same basic mechanism as traditional radical
polymerization, which is a three-step chain reaction whose steps are initiation, propagation, and
termination (Figure 2). Initiation is the step in which the polymer chain begins growing. In this
step, radicals are generated and added to a monomer unit. Propagation is the step in which most
of the chain’s growth occurs. It involves the repeated addition of the growing radical polymer to
the alkene monomer molecules. Each propagation reaction results in a radical chain that is one
repeat unit longer. Termination is the step in which the chain stops growing, and it can occur in
several different ways. Two of the possibilities include radical combination, which will decrease
the concentration of radicals in the reaction mixture, and transfer of the radical to another
molecule, which will not decrease the radical concentration.
I2
kd
2I
Heat or
Light
Initiation
ki
I
I
+
Y
Y
kp
I
Y
I
+
n
Y
n+1
Y
Y
Y
Y
Propagation
Y
(Pn )
Pn
Pn
Pn
Z
S
S
S
C
C
C
S
Z
R
S
Z
R
ki
I
+
R
Y
Reinitiation
Y
Pn
Pn
Pn
Y
Z
S
S
S
C
C
C
S
Z
Pm
Chain transfer
to RAFT agent
R
S
S
Z
Pm
Pm
Chain
equilibrium
S
Y
Propagation
Propagation
Pn
+
Pm
dead polymer
Termination
Figure 2. Mechanism of reversible addition-fragmentation chain transfer polymerization
3
In order to obtain low molecular weight distribution (MWD) or polydispersity, it would be ideal
to start all polymerizations at the same time, thus allowing all of the chains to grow under
identical conditions. However, with traditional radical polymerization, the rate of radical
termination is so high that most of the chains would barely grow at all before termination.
Usually with this method, it is necessary to constantly add initiator in order to offset termination
and keep the reaction going. In order to alleviate this problem, it is possible to cap the end of
each chain with a non-radical group that can be easily removed to give a radical. Ideally, the
majority of the chains would be capped (and thus dormant) at any one time. Since the end group
would be easily removed, the objective would be to obtain equilibrium between the dormant and
active chains, thus allowing all of the chains the same opportunity to propagate.
There are several types of capping groups, all of which operate through different mechanisms.
RAFT polymerizations in particular utilize what is called a degenerative transfer mechanism in
which the activation-deactivation process involves the reaction of polymer chains that transfer
the capping group back-and-forth to each other. These chains are typically identical in all but
number of repeat units. The mechanism gets its name because the overall exchange of the
capping group is energetically degenerate. RAFT polymerizations are the most successful type
of polymerization using this mechanism, and they involve dithio-based esters, carbamates, and
xanthates. During the early stages of a RAFT polymerization, radicals are generated through the
decomposition of the radical initiator. These radicals will propagate a few times and then react
with what is called the “RAFT agent” which gets passed between chains, keeping the
polymerizations “living” [6].
Proposed Work
The synthesis of poly(methacrylic acid) is difficult in that attempting to polymerize methacrylic
acid directly often results in by-products and difficult purification and characterization
procedures. To avoid this problem, the polymer will be synthesized by first polymerizing the
“protected” methacrylate monomer tert-butyl methacrylate (t-BMA) using RAFT polymerization
techniques and then removing the dithioester end group, to give a thiol. The protecting groups
can then be removed using acid hydrolysis. This procedure will be completed to form
monodisperse polymers with number average molecular weights (Mn) equaling approximately
2,000, 4,000, 7,500, and 15,000. Preliminary results show that the synthesis of these polymers
should be possible. Molecular weights and polydispersities will be determined using gel
permeation chromatographic methods (GPC).
O
O
HO
OH
Methacrylic Acid
O HO
O
O HO
O
Poly(methacrylic acid)
Figure 3. Polymerization of methacrylic acid
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t-BMA
Fluorescence spectroscopy will be used to study the effects of molecular weight and
polydispersity on the conformational transition of the polymer samples. Pyrene has been used in
previous studies as a probe for both poly(methacrylic acid) and poly(2-ethacrylic acid) and will
be used here as well [4]. The intensities of the peaks at 373 nm and 384 nm on the emission
spectrum generally increases in nonpolar environments, and the ratio between these values thus
varies with the polarity of the environment. The width of the peaks will also be analyzed to
study the breadth of the conformation changes and how it correlates to the polydispersity of the
sample [4].
Preliminary Results
Initial experiments have been performed to determine whether the desired product can be
synthesized using the proposed methods. The synthesis of poly(t-BMA) was very successful,
and Figure 4 shows the overall polymerization and resulting polymer. The overall conversion of
monomer to polymer was 31%, which is relatively low. However, this is due to the goal of
obtaining low molecular weight polymers with high end group fidelity.
O
S
S
CH3
O
CN
S
+
O
CH3
AIBN, 60°C
OR
S
Anisole
C
CH2
C
O
DP05
C
CH3
CN
O
O
H3 C
C
Mn = 5,100
Pd = 1.18
n
CH3
CH3
Figure 4. Overall polymerization of t-BMA
Two post-polymerization reactions were performed on the poly(t-BMA): the first (DP15) was
removal of the dithiobenzoate end group to give a thiol end group; the second (DP07) was
deprotection of the t-butyl group to give the carboxyl acid group. These reactions are
summarized in Figure 5.
5
S
CH3
S
C
CH2
n
C
C
CH3 H N
2
NH2
CHCl3, 40°C
CN
O
DP05
CH3
CH3
O
HS
C
CH3
CH2
n
C
O
C
CH3
CN
O
DP15
1,4 dioxane
H2O, HCl
reflux
reflux
1,4 dioxane
H2O, HCl
CH3
S
CH3
S
C
CH2
C
DP07
O
HS
CH3
n
C
C
CH3
CH2
C
CH3
O
CN
n
C
CH3
CN
OH
Desired Product
OH
Figure 5. Reactions of poly(t-BMA)
The product from the reaction removing the dithiobenzoate end group (DP15) was analyzed
using GPC and proton NMR. The GPC results show a molecular weight of 6,200 and a
polydispersity of 1.21. This increase in molecular weight is most likely due to calibration error.
It should be noted that GPC calibration normally has approximately 10% error. A change from
5,100 to 6,200 (~ 20%) observed here is slightly larger than the usual error and is most probably
due to inconsistent calibration. In addition, the polymer underwent a change of color from pink
to white, which suggests that the reaction was a success. The proton NMR spectrum of the
product backs this up.
The product from the deprotection reaction (DP07) was analyzed using solubility tests in water,
chloroform, tetrahydrofuran (THF), and dimethylsulfoxide (DMSO). The compound was found
to be soluble in water and DMSO, but not in THF and chloroform. These solubility results
indicate that the polymer was successfully deprotected, as the removal of the protecting group
would have changed its polarity. For more results, see Synthesis of Poly(Methacrylic Acid)
Using Reversible Addition-Fragmentation Chain Transfer (RAFT) Polymerization [7].
Timetable
Date
May 2005
Goals
Experiment with new synthesis technique: in
vivo RAFT polymerization
Synthesize several monodisperse
poly(methacrylic acid) samples with molecular
weights in the range of 2,000 to 15,000
Study the effects of molecular weight and
polydispersity on conformational transition of
Fall 2005
Spring 2005
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poly(methacrylic acid) in aqueous solution
using fluorescence spectroscopy, and write and
revise thesis.
Overview
Poly(carboxylic acid)s have uses in both industry and the biological arena. These compounds’
pH-induced rod-to-globule conformational transition allows for their integration into lipid
membranes to make them environmentally responsive. The resulting polymer-lipid membranes
have possible uses in gene therapy, targeted drug delivery, etc. In order for the polymer to be
effective for these purposes, the “critical pH” must be well-defined. Studies on the
conformational transition of PEAA have shown that monodisperse low molecular weight PEAA
samples offer better definition of the “critical pH” than polydisperse high molecular weight
samples. The pH-induced conformational changes of PMA, a similar compound to PEAA, have
not yet been researched using monodisperse low molecular weight polymers, and this topic will
be the focus of the proposed thesis.
The synthesis of low molecular weight, monodisperse PMA will be performed by polymerizing
the “protected” methacrylate monomer tert-butyl methacrylate (t-BMA) using the LRP technique
of RAFT polymerization. The dithioester end group and protecting groups will then be removed
from the polymer in subsequent reaction steps. Preliminary results show that the synthesis of
these polymers should be possible. Fluorescence spectroscopy, using pyrene as a probe, will be
utilized to study the effects of molecular weight and polydispersity on the conformational
transition of the polymer samples. It is hypothesized that monodispersed low molecular weight
PMA will have a well-defined “critical” pH.
Literature Cited
[1]
Olea, A.F. and Thomas, J.K. Fluorescence Studies of the Conformational Changes of
Poly(methacrylic acid) with pH. Macromolecules 1989, 22, 1165 – 1169.
[2]
Tirrell, D.A., et. al. H+-Induced Release of Contents of Phosphatidylcholine Vesicles
Bearing Surface-Bound Polyelectrolyte Chains. J. Am. Chem. Soc. 1988, 110, 7455 –
7459.
[3]
Linhardt, J.G., and Tirrell, D. pH-Induced Fusion and Lysis of Phosphatidylcholine
Vesicles by the Hydrophobic Polyelectrolyte Poly(2-ethacrylic Acid), Langmuir 2000.
[4]
Linhardt, J., Thomas, J., and Tirrell, D. Free-Radical Synthesis of Poly(2-ethacrylic acid)
Fractions of Low Polydispersity: Effects of Molecular Weight and Polydispersity on the
pH-Dependent Conformational Transition in Aqueous Solutions. Macromolecules 1999,
32, 4457 – 4459.
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[5]
Olea, A.F., Rosenbluth, H., and Thomas, J.K. Effect of the Molecular Weight on the
Dynamics of the Conformational Transition of Poly(methacrylic acid). Macromolecules
1999, 32, 8077 – 8083.
[6]
Chiefari, J., et. al. Living Free-Radical Polymerization by Reversible AdditionFragmentation Chain Transfer: The RAFT Process, Macromolecules 1998, 31, 5559.
[7]
Petko, D., and Shipp, D. Synthesis of Poly(Methacrylic Acid) using Reversible
Addition-Fragmentation Chain Transfer (RAFT) Polymerization.
Symposium for
Undergraduate Research Experiences Summer 2004.
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