Thiol-ene click functionalization and subsequent polymerization of 2-oxazoline monomers

Article abstract of DOI:10.1021/ma9028294

Polymers of 2-oxazolines are a class of polymers that can exhibit water solubility and biocompatibility depending upon the N-acyl side-chain functionality and can be readily modified at both the end-groups and backbone, making them appealing alternatives

Full text of DOI:10.1021/ma9028294

Macromolecules 2010, 43, 4081–4090 4081
DOI: 10.1021/ma9028294
Thiol-Ene Click Functionalization and Subsequent Polymerization
of 2-Oxazoline Monomers
Mallory A. Cortez and Scott M. Grayson*
Department of Chemistry, Tulane University, New Orleans, Louisiana 70118
Received December 23, 2009; Revised Manuscript Received March 16, 2010
ABSTRACT: Polymers of 2-oxazolines are a class of polymers that can exhibit water solubility and
biocompatibility depending upon the N-acyl side-chain functionality and can be readily modified at both
the end-groups and backbone, making them appealing alternatives to poly(ethylene glycol) (PEG). Methods
to introduce a range of functionality onto oxazoline monomers are of interest due to the limited availability of
functionalized oxazoline monomers. Prepolymerization thiol-ene coupling between a side-chain thiol and
2-isopropenyl-2-oxazoline was used to synthesize three different monomers followed by polymerization to
yield 2-substituted oxazoline polymers with aryl, ester, protected amine, and protected carboxylic acid side
chains. The resulting polymers exhibited molecular weights ranging form 3000 to 8000 and polydispersities
typically below 1.3. The deprotection of these polymers was also achieved to yield polymers with reactive
functionalities, including a cysteine with either the amine or carboxylic acid exposed on each repeat unit.
Introduction
2-oxazolines can follow either ionic or covalent mechanisms
depending on the relative nucleophilicity of the counteranion
The general cationic ring-opening polymerization reaction of
2-oxazolines follows a living mechanism that leads to well-
defined poly(N-acylethylenimine) polymers (PNAIs) with low
polydispersities.1 The ease with which the backbone and end-
groups of poly(oxazolines) can be modified makes this family of
polymers attractive for broad applications.2-4 In addition, the
high water solubility5-7 and biocompatibility of the short side-
chain derivatives of 2-oxazolines (2-methyl- and 2-ethyl-) make
these N-acyl derivatives attractive alternatives to PEG.2-4 For
example, Zalipsky and co-workers8,9 have demonstrated that the
replacement of PEG with poly(2-methyl-2-oxazoline) or poly-
(2-ethyl-2-oxazoline) provided similar advantages for polymer-
modified liposomes, including increased plasma lifetimes,
decreased blood clearance, and increased solubility in organic
solvents. PNAIs have also been successfully used as a component
in amphiphilic polymer micelles for the encapsulation of Doxor-
ubicin,10 as polymer-drug conjugates for high and low molecular
weight biomolecules such as trypsin and Ara-C,11 as 111In-
enriched scaffolds for radiotherapy and tumor imaging,12 as non-
ionic polymer surfactants, and as the hydrophilic component of
cross-linked hydrogels.1
In contrast to PEG, which only has the capacity for end-group
functionalization, the N-acyl side chain of oxazoline polymers
enables diverse functionalization along the polymer backbone.
As a result, the chemical properties and solubility of these
polymers are readily tunable based on the functionality of the
side chain at the substituted 2-position.1,3,13 Though a number of
2-substituted oxazolines are commercially available, the modular
syntheses of oxazolines containing a wider range of functional
side chains would be advantageous for both biomedical and
materials science applications.
derived from the initiator versus the monomer. Initiators with
triflate or tosylate counterions tend to follow the ionic route while
halide initiators propagate using a covalent intermediate.14
Termination occurs following the addition of a strong nucleo-
phile or adventitious reactions with water. This versatility in
initiation and termination allows for the introduction of different
functionalities at either end of the polymer chain.
Side-chain functionality can be introduced to a polymer either
by attachment of the desired functionality to the monomer before
polymerization or by postpolymerization modification of a
reactive functionality on the polymer backbone.13,15-18 Post-
polymerization functionalization of the repeat units can enable
access to functional PNAIs with high degrees of polymerization;
however, this route can suffer from incomplete side-chain mod-
ification. In particular, thiol-ene chemistry has been used in the
postpolymerization modifications of poly(oxazolines) as re-
ported by Schlaad and co-workers to tune the LCSD properties
of polyoxazoline.13 On the other hand, the direct polymerization
of modified oxazoline monomers should exhibit quantitative
functionalization, however, and requires protection of side-chain
functionalities that are incompatible with the cationic ring-
opening polymerization. In addition, this route is expected to
be prone to steric retardation of polymerization rates, resulting in
lower degrees of polymerization. Because only a limited diversity of
functionalized oxazoline monomers are commercially available,
thiol-ene coupling between the readily available 2-isopropenyl-
2-oxazoline and a library of thiols was explored as a route toward a
wider range of oxazoline monomers, and their resultant poly-
merizations reactions were investigated.
The utilization of highly efficient “click reactions”, as termed
by Kolb et al.,19 has been widespread due to their high specificity,
near-quantitative yields, and near-perfect fidelity in the presence
of most functional groups.20 The Cu(I)-catalyzed [3 þ 2] cyclo-
addition reaction between an azide and an alkyne has been the
most utilized of the click reactions because it is fast, high-yielding,
functional group tolerant, and compatible with a range of
The polymerization of cyclic imino ethers is thermodynami-
cally favored due to the favorable isomerization of the imino
ether group to the amide functionality and elimination of mono-
mer ring strain. The cationic ring-opening polymerization of
*Corresponding author. E-mail: sgrayson@tulane.edu.
r 2010 American Chemical Society
Published on Web 04/08/2010
pubs.acs.org/Macromolecules

4082 Macromolecules, Vol. 43, No. 9, 2010
Cortez and Grayson
solvents. However, this type of click reaction is complicated by
the Cu catalyst, which complexes strongly to electron-donating
functionalities (such as amines, thiols, alcohols, etc.) and can
result in unacceptable levels of contamination (single ppms) for
both biological and materials applications.21 Different methods
have been investigated to overcome this complication, such as
catalyst-free triazole couplings;22,23 however, the strained or
fluorinated alkyne substrates require substantial synthetic effort
and the absence of a catalyst offers limited control over coupling.
Recently, the addition of thiols to alkenes (thiol-ene) has
emerged as an attractive complementary and metal-free click
process.24,25 This reaction can be catalyzed photochemically or
by nucleophiles such as amines depending on the electrophilicity
of double bond with near-quantitative yields in a period of
seconds at ambient temperatures while showing compatibility
with a wide range of functional groups in a variety of different
solvents.24-28 The high efficiency of the thiol-ene reaction has
enabled the preparation of well-defined films of uniform molec-
ular networks exhibiting high glass transition temperatures as
well as highly cross-linked networks.24,26,27 Recently, many
examples of the thiol-ene reaction in other areas of material
science have been reported,29 including the syntheses of dendri-
mers30 and star polymers26 and functionalization of polymer end-
groups and surfaces.13,15,26,31 This type of chemistry has also been
studied for use in biomedical and pharmaceutical applications32
including the synthesis of hydrogels33,34 and controlled drug
release systems.35 In addition, postpolymerization modifications
of poly(oxazolines) using thiol-ene reactions have been per-
formed by Schlaad and co-workers.13 The absence of the Cu
catalyst makes the thiol-ene coupling attractive for biological
applications and therefore was applied toward the preparation of
functionalized 2-substituted oxazoline monomers.
solutions of 1,8,9-anthracenetriol matrix (20 mg mL-1) in
chloroform, NaTFA (2 mg mL-1) as the counterion in THF,
and 2 mg mL-1 of sample in THF were used. MALDI samples
were then prepared by using a matrix solution/counterion
solution/sample solution ratio of 1:1:1. The pulsed ion extrac-
tion delay was set to 0 ns, with ion source 1 voltage of 19.00 kV
and ion source 2 voltage of 16.47 kV, a lens voltage of 8.30 kV, a
low mass gate at 800 amu, and a laser power set to 55%. For
polymer 5, solutions of 1,8,9-anthracenetriol matrix (20 mg
mL-1) in chloroform, NaTFA (2 mg mL-1) as the counterion
in THF, and 2 mg mL-1 of sample in THF were used. MALDI
samples were then prepared by using a matrix solution/counter-
ion solution/sample solution with a ratio of 1:2:1. The pulsed ion
extraction delay was set to 50 ns, with ion source 1 voltage of
19.00 kV and ion source 2 voltage of 16.70 kV, a lens voltage of
6.40 kV, a low mass gate at 600 amu, and a laser power set to
31%. For polymer 6, solutions of galvinoxyl, free radical matrix
(10 mg mL-1) in THF, NaTFA (2 mg mL-1) as the counterion in
THF, and 2 mg mL-1 of sample in THF were used. MALDI
samples were then prepared using a matrix solution/counterion
solution/sample solution with a ratio of 1:0.7:1. The pulsed ion
extraction delay was set to 0 ns, with ion source 1 voltage of
19.00 kV and ion source 2 voltage of 8.30 kV, a lens voltage of
8.30 kV, a low mass gate at 800 amu, and a laser power set to
55%. Number-average molecular weight (Mn) and polydisper-
sity (PDI) were calculated using PolyTools software.
Gel permeation chromatography (GPC) was carried out on a
Waters model 1515 series pump (Milford, MA) with three-
column series from Polymer Laboratories, Inc., consisting of
PLgel 5 μm Mixed D (300 mm ꢀ 7.5 mm, molecular weight
˚
range 200-400 000), PLgel 5 μm 500 A (300 mm ꢀ 7.5 mm,
˚
molecular weight range 500-30 000), and PLgel 5 μm 50 A
(300 mm ꢀ 7.5 mm, molecular weight range up to 2000)
columns. The system was fitted with a model 2487 differential
refractometer detector, and anhydrous tetrahydrofuran was
used as the mobile phase (1 mL min-1 flow rate) at 25 °C. The
resulting molecular weight was based on calibration using linear
polystyrene standards.
Herein, we report the functionalization and polymerization of
three different 2-substituted oxazoline monomers utilizing the
amine-catalyzed thiol-ene click reaction between the commer-
cially available 2-isopropenyl-2-oxazoline and three thiols: thio-
Infrared (IR) spectroscopy was implemented using a NEXUS
670 FT-IR ESP (Madison, WI). Samples were made using
∼4 mg of polymer and 5 mg of KBr which was ground into a
fine powder by mortar and pestle and compacted into a pellet.
Proton (and carbon) nuclear magnetic resonance (NMR)
spectra were obtained from a 400 MHz (100 MHz) Varian
Mercury spectrometer (Palo Alto, CA), using TMS = 0.00 ppm
calibration and a 500 MHz (125 MHz) Bruker spectrometer
(Billerica, MA), using TMS = 0.00 ppm calibration with deuter-
ated solvents at room temperature.
Synthesis of Butylamine Resin.36 Butylamine (29.26 g,
400 mmol) was added to 75 mL of DMF in a round-bottom
flask containing a magnetic stir bar. Merrifield’s peptide resin
(20 g, 80 mmol) was then added to the reaction flask and
refluxed overnight. The solid resin was then filtered and washed
with 250 mL of dichloromethane (DCM) until no unreacted
butylamine remained. The butylamine resin was then dried
under vacuum overnight, and an IR was taken.
General Procedure for the Functionalization of 2-Isopropenyl-
2-oxazoline Monomer. 2-Isopropenyl-2-oxazoline (1.06 g,
9.078 mmol) was dissolved in 5 mL of dry tetrahydrofuran
(THF) in a round-bottom flask with a magnetic stir bar. The
desired thiol (e.g., thiophenol 1.002 g, 9.078 mmol) was added,
and the reaction mixture was stirred under N2 gas for 5 min. The
butylamine resin (1.54 g, 4.7896 mmol) was then added, and
the reaction was allowed to stir overnight at room temperature.
The butylamine resin was filtered off and washed with DCM.
The filtrate was concentrated, characterized, and used without
further purification (yield: 95%). Representative characteriza-
tion (racemic) data for thiophenol (1) follow.
phenol, methyl thioglycolate, and N-(tert-butoxycarbonyl)-L-
cysteine methyl ester.
Experimental Section
Materials. Merrifield’s peptide resin (1% cross-linked) was
purchased from Sigma-Aldrich (St. Louis, MO). Acetonitrile
(reagent grade, Fisher Scientific) was purified by distillation at
85 °C and placed over molecular sieves until used. Methyl
tosylate (98%) was purified by distillation at 140 °C and placed
over molecular sieves until used. 2-Isopropenyl-2-oxazoline
(g99%), thiophenol (97%), methyl thioglycolate (95%),
N-(tert-butoxyarbonyl)-L-cysteine methyl ester (97%), butyl-
amine (99.5%), trifluoroacetic acid (99%), and sodium trifluoro-
acetate (98%) were purchased from Aldrich and used without
further purification. 1,8,9-Anthracenetriol (puriss. for MALDI-
MS) was purchased from Fluka and used without further
purification. Ammonium hydroxide (reagent grade) was pur-
chased from Fisher Scientific and used without further purifica-
tion. All other solvents were reagent grade, purchased from
Fisher Scientific, and used without further distillation or pur-
ification.
Instrumentation. Mass spectral data were acquired using a
Bruker Autoflex III (Billerica, MA) matrix-assisted laser de-
sorption ionization time of flight mass spectrometer (MALDI)
with delayed extraction using the reflector and positive ion
mode. Mass spectral data were also obtained with a Bruker
MicrOTOF electrospray ionization (ESI) mass spectrometer
with an Agilent 1200 series binary pump (Santa Clara, CA).
Gas chromatography-mass spectrometry (GCMS) was car-
ried out on a Varian 450-GC gas chromatograph with a Varian
300-MS SQ mass spectrometer (Palo Alto, CA). For polymer 4,
Characterization of 1. The monomer 1 was isolated as its
racemate (yield: 95%). 1H NMR (CDCl3, δ, ppm): 1.30 (d, 3H,

Article
Macromolecules, Vol. 43, No. 9, 2010 4083
J = 6.8 Hz, (-CH3)), 2.70 (sx, 1H, J = 6.96 Hz (-Coxazoline
-
J = 7.6 Hz, 13.4
follow. 1H NMR (CDCl3, δ, ppm): 1.0-1.4 (br (-CH3)),
2.6-2.8 (br (-CHCH2S-)), 2.8-3.1 (br (-CCHCH3CH2-)),
CH(CH3)CH2-)), 2.934 (dd, 1H,
(-CHCHaHbS-)), 3.29 (dd, 1H, J1 = 6.4 Hz, J2=13.2 Hz
(-CHCHaHbS-), 3.77 (t, 2H, J = 9.6 Hz (-NCH2CH2O-)),
4.13 (t, 2H, J = 9.6 Hz (-NCH2CH2O-)), 7.16 (m, 1H, J = 7.2
Hz (Ar(H)), 7.26 (m, 2H, J = 7.0 Hz (Ar(H)), 7.35 (m, 2H, J =
6.0 Hz (Ar(H)). 13C NMR (CDCl3, δ, ppm): 18.5 (-CH3), 33.0
(-CoxazolineCHCH2S-), 38.0 (-CHCH2S-), 54.0 (-NCH2-
CH2O-), 68.0 (-NCH2CH2O-), 126.0 (Ar(C)), 129.0
(Ar(C)), 131.0 (Ar(C)), 136.0 (Ar(C)), 170.0. (-NdC-). IR:
1600 (Ar(CdC)), 1670 (NdC), 3080 (Ar(C-H)), 3050
(Ar(C-H).
Characterization of 2. The monomer 2 was isolated as its
racemate (yield 90%). 1H NMR (CDCl3, δ, ppm): 1.25 (d, 3H,
J = 6.4 Hz (-CH3)), 2.725 (dd, 2H, J1 = 6.4 Hz, J2 13.6 Hz
(-CHCH2S-)), 2.94 (m, 1H, J =6.56 (CoxazolineCHCH3CH2-)),
3.23 (s, 2H (-SCH2COOC3)), 3.71, (s, 3H (-COOCH3)), 3.82 (t,
2H, J = 9.6 Hz (-NCH2CH2O-)), 4.22 (t, 2H, J = 9.2 Hz
(-NCH2CH2O-). 13C NMR (CDCl3, δ, ppm): 17.0 (-CH3),
34.0 (-CoxazolineCHCH2S-), 34.0 (-CHCH2S-), 37.0 (-SCH2-
COOCH3), 52.0(-COOCH3), 54.0 (-NCH2CH2O-), 67.5
(-NCH2CH2O-), 170.0 (-NC-), 171.0 (-CH2COOCH3). IR:
1250 (ester (O-C)), 1670 (NdC), 1740 (CdO).
Characterization of 3. The monomer 3 was isolated as its
racemate (yield 97%). 1H NMR (CDCl3, δ, ppm): 1.23 (d, 3H,
J = 6.28 Hz (-CH3)), 1.425 (s, 9H (-OC(CH3)3)), 2.64 (m, 2H
(CoxazolineCHCH2S-)), 2.84 (m, 1H, (CoxazolineCHCH3CH2-)),
2.95 (d, 2H, J = 4.8 Hz (-SCH2CH-)), 3.70 (s, 3H
(-COOCH3)), 3.82 (t, 2H, J = 9.6 Hz (-NCH2CH2O-)),
4.22 (t, 2H, J = 9.6 Hz (-NCH2CH2O-)), 4.51 (m, 1H
(-CH2-NHCHCOOCH3). 13C NMR (CDCl3, δ, ppm): 17.0
(-CH3), 28.0 (-OC(CH3)3), 34.0 (-CoxazolineCHCH3CH2S-),
35.0 (-CoxazolineCHCH3CH2S-), 37.0 (-SCH2CH), 52.5
(-COOCH3), 54.0 (-SCH2CHCOOCH3), 54.3 (-NCH2-
CH2O-), 67.5 (-NCH2CH2O-), 80.0 (-NHCOOC(CH3)3),
155.5 (-NHCOOC-), 170.0 (-NC-), 172.0 (-CCOOCH3).
IR: 1250 (ester (O-C)), 1670 (NdC), 1720 (CdO), 1760
(carbamate (O = C)), 3200 (N-H), 3350 (N-H).
General Procedure for the Polymerization of Functionalized 2-
Isopropenyl-2-oxazoline. The desired 2-isopropenyl-2-oxazoline
functionalized monomer was dried under vacuum overnight to
remove any water. All glassware was flame-dried. Distilled
acetonitrile (4-6 mL) was added to the reaction flask under
N2 gas with a syringe. Varying initiator to monomer ratios were
studied including 1:50, 1:100, 1:150, and 1:200. In this example,
an initiator to monomer ratio of 1:100 was employed. Distilled
methyl tosylate (0.0126 g, 0.0678 mmol) was added to the
reaction via syringe. Functionalized 2-isopropenyl-2-oxazoline
monomer (e.g., monomer 1, 1.5 g, 6.785 mmol) was added
via syringe, and the reaction was stirred for 24 h at 70 °C. After
24 h the reaction was cooled in an ice bath, and 0.5 mL of H2O
was added and stirred overnight at room temperature.
The polymer was precipitated into diethyl ether and filtered.
The polymer was washed with NaHCO3 to remove any tosylate
anion (conversion: 19%).
3.1-3.8 (br (-NCH2CH2-)), 6.9-7.4 (br (CH from (Ph))). 13
C
NMR (CDCl3, δ, ppm): 19 (-CH3), 37 (-CCHCH3CH2-), 38
(-CHCH2S-), 48 (-NCH2CH2-), 126 (Ar(C)), 137 (Ar(C)),
139 (Ar(C)), 146 (Ar(C)), 186 (-NCOCH-). IR: 1600
(Ar(CdC)), 1640 (tertiary amide(C-N)), 3050 (Ar(C-H),
3080 (Ar(C-H)), 3300 (O-H), 3500 (O-H).
Characterization of 5. MALDI-TOF MS was taken at a
matrix/counterion/sample ratio of 1:2:1, with 1,8,9-anthracene-
triol matrix (20 mg/mL), NaTFA (2 mg/mL) as the counterion,
and 2 mg/mL of sample. Representative characterization data
follow. 1H NMR (CDCl3, δ, ppm): 1.10-1.30 (br (-CH3)),
2.55-2.70 (br (-CHCH2S-)), 2.85-3.10 (br (-CCHCH3CH2-)),
3.10-3.30 (br (-SCH2C-)), 3.40-3.60 (br (-NCH2CH2-))
3.60-3.75 (br (-COOCH3)). 13C NMR (CDCl3, δ, ppm): 19
(-CH3), 34 (-CCHCH3CH2-), 36 (-CCH2S-), 37 (-SCH2CO-
OCH3), 46 (-NCH2CH2-), 52 (-COOCH3), 172 (-CH2COO-
CH3), 176 (-NCOC-). IR: 1640 (tertiary amide (C-N)), 1735
(ester (CdO)), 3500 (O-H), 3200 (O-H).
For the purification of polymer 5f (Table 1), a different work-
up was employed to isolate the highest molecular weight fraction
of the polymer. The above-described polymerization procedure
was employed with a 1:130 initiator (0.0132 g, 0.0709 mmol) to
monomer (monomer 2, 2 g, 9.2133 mmol) ratio with acetonitrile
as solvent (4 mL) followed by the addition of H2O (0.5 mL),
precipitation in diethyl ether, filtration, and wash (conversion:
14%); Mn (GPC, before collection of high molecular weight
fraction): 4000. The polymer (0.1500 g, 0.0375 mmol) was then
dissolved in toluene (150 mL) with a small amount of dichlor-
omethane (25 mL). Diethyl ether was then added dropwise until
cloudy (∼50 mL). The solution was then heated using a hot air
gun until clear and allowed to precipitate overnight overnight.
The precipitate was then collected. This was repeated twice
(overall yield: 4.8%).
Characterization of 6. MALDI-TOF MS was taken at a
matrix/counterion/sample ratio of 1:0.7:1, with galvinoxyl, free
radical (10 mg/mL) matrix, NaTFA (2 mg/mL) as the counter-
ion, and 2 mg/mL of sample. Representative characterization
data follow. 1H NMR (CDCl3, δ, ppm): 1.00-1.20 (br (-CH3)),
1.25-1.50 (br (-C(CH3)3)), 2.40-2.65 (br (-CHCH2S-)),
2.65-3.10 (br (-CCHCH3CH2-)); (-SCH2CH-), 3.40-3.60
(br (-NCH2CH2-)), 3.61-3.78 (br (-COOCH3)), 4.35-4.55
(br (-SCH2CH-)). 13C NMR (CDCl3, δ, ppm): 19.0 (-CH3),
29.0 (C(CH3)3), 35.0 (-CHCH2S-), 37.0 (-CHCH2S-), 39.0
(-SCH2CH-), 44.0 (-NCH2CH2-), 52.0 (-COOCH3), 54.0
(-SCH2CH-), 81.0 (-COOC(CH3)3), 156.0 (-NHCOOC-
(CH3)3), 171.5 (-CHCOOCH3-), 177.0 (-NCOOCH-). IR:
1640 (tertiary amide (-N-)), 1710 (C-O), 1760 (carbamate
(OdC)), 3350 (O-H, N-H), 3200 (O-H, N-H).
Deprotection of Carboxylic Acid Functionality of 5. Ammo-
nium hydroxide (3 mL) was added to methyl thioglycolate
functionalized 2-isopropenyl-2-oxazoline polymer (25 mg,
5.6818 mmol) in a 5 mL vial. The vial was then capped and heated
in a sand bath at 90 °C overnight. The ammonium hydroxide and
any water present were then evaporated, and the sample was
further dried under vacuum. Representative characterization data
follow. 1H NMR (D2O, δ, ppm): 1.20-1.26 (br (-CH3)),
2.60-2.80 (br (-CHCH2S-)), 2.80-2.90 (br (-CCHCH3CH2-)),
3.00-3.20 (br (-SCH2C-)), 3.20-3.60 (br (-NCH2CH2-)). 13C
NMR (CD3OD, δ, ppm): 19.0 (-CH3), 36.0 (-CCHCH3CH2-),
37.0 (-CCH2S-), 37.5 (-SCH2COOCH3), 48.0 (-NCH2CH2-),
175.0 (-CH2COOH), 178.5 (-NCOC-). IR: 1640 (tertiary amide
(C-N)), 1660 (ester (CdO)), 3500 (O-H), 3200 (O-H), 3310
(O-H).
For the determination of monomer conversion versus molec-
ular weight, the polymerization procedure described above was
employed with a 1:130 initiator (0.0129 g, 0.0696 mmol) to
monomer (monomer 1, 2 g, 9.0462 mmol) ratio with acetonitrile
as solvent (4 mL). The reaction was heated to 65 °C. Aliquots
(0.25 mL) of the bulk reaction were removed every 2 h with a
disposable syringe. The samples were cooled in an ice bath, and
0.1 mL of H2O was added. The sample was then concentrated to
remove acetonitrile with a rotary evaporator. No further pur-
ification was preformed on the samples before acquiring GPC
data (Figure 7).
Deprotection of Carboxylic Acid Functionality of 6. Ammo-
nium hydroxide (3 mL) was added to N-(tert-butoxycarbonyl)-
L-cysteine methyl ester functionalized 2-isopropenyl-2-oxazo-
line polymer (25 mg, 3.5714 mmol) in a 5 mL vial. The vial
was then capped and heated in a sand bath at 90 °C overnight.
Characterization of 4. MALDI-TOF MS was taken at a
matrix/counterion/sample ratio of 1:1:1, with 1,8,9-anthracene-
triol matrix (20 mg/mL), NaTFA (2 mg/mL) as the counterion,
and 2 mg/mL of sample. Representative characterization data

4084 Macromolecules, Vol. 43, No. 9, 2010
Cortez and Grayson
Table 1. Initiator to Monomer Ratio (I:Mono), Monomer Concentration ([Mono]), Temperature, Time, Number-Average Molecular Weight (Mn),
Polydispersity Index (PDI = Mn/Mw), and Degrees of Polymerization (DP = Mn/[Mass Repeat Unit]) Data for Different Trials of the Three
Functionalized Oxazoline Polymer Samplesa
Mn
MALDI
PDI
MALDI
DP
MALDI
polymer/trial
I:Mono
[Mono], mmol
temp (°C)
time (h)
GPC
GPC
GPC
thiophenol-functionalized 2-isopropenyl-2-oxazoline polymer (4)
4a
4b
4c
4d
4e
1:100
1:150
1:150
1:150
1:150
6.7850
13.5693
11.3070
11.3078
11.3078
70
80
90
70
70
24
48
48
48
36
4300
3800
3800
3900
4000
4400
3600
4300
4300
4200
1.17
1.15
1.16
1.17
1.17
1.11
1.10
1.07
1.07
1.23
19
17
17
18
18
20
16
19
19
19
methyl thioglycolate -functionalized 2-isopropenyl-2-oxazoline polymer (5)
5a
5b
5c
5d
5e
1:50
9.2133
6.9099
7.3706
11.5176
13.8198
65
70
70
70
80
24
24
24
48
48
3700
3800
3900
4700
5000
4000
4200
4600
4500
4400
1.31
1.15
1.16
1.24
1.29
1.11
1.11
1.13
1.16
1.13
16
17
18
22
23
18
19
21
21
20
1:100
1:100
1:150
1:150
5f
1:130
9.2133
70
48
9100
n/a
1.27
n/a
42
n/a
N-(tert-butoxycarbonyl)-L-cysteine methyl ester-functionalized 2-isopropenyl-2-oxazoline polymer (6)
6a
6b
6c
6d
1:50
3.7556
13.7510
7.2159
7.2159
70
80
70
90
24
48
24
24
4000
4700
5000
2800
n/a
n/a
n/a
n/a
1.18
1.32
1.30
1.01
n/a
n/a
n/a
n/a
11
13
14
8
n/a
n/a
n/a
n/a
1:100
1:100
1:200
a Polymer trial 5f was synthesized using the same procedure as trails 5a-5e, followed by a separation of the highest molecular weight fraction as
described in Experimental Section. For numbering of polymers, see Scheme 1.
Scheme 1. Synthetic Scheme of the Functionalization and Polymeri-
zation of 2-Isopropenyl-2-oxazolinea
The ammonium hydroxide and any water were then evaporated,
and the sample was then further dried on a vacuum pump.
Representative characterization data follow. 1H NMR (D2O): δ
0.9-1.00 (b (-CH3)), 2.4-2.56 (b (-C(CH3)3)), 2.58-2.72 (b
(-CHCH2S-)), 3.02-3.06 (b (-CCHCH3CH2-)); (-SCH2-
CH-), 3.2-3.6 (b (-NCH2CH2-)), 4.05 (br (-SCH2CH-)).
13C NMR (CD3OD):
δ 19 (-CH3), 29 (C(CH3)3), 35
(-CCHCH3CH2S-), 37 (-CHCH2S-), 38 (-SCH2CH-), 46
(-NCH2CH2-), 52 (-SCH2CH-), 80 (-COOC(CH3)3), 157
(-NHCOOC(CH3)3), 176 (-CHCOOH), 178 (-NCOOCH-).
IR: 1640 (tertiary amide (C-N)), 1710 (CdO), 1760 (carbamate
(CdO)), 3350 (O-H, N-H), 3200 (O-H, N-H).
Deprotection of Amine Functionality of 6. N-(tert-Butoxy-
carbonyl)-L-cysteine methyl ester functionalized 2-isoprope-
nyl-2-oxazoline (25 mg, 3.5724 mmol) was either stirred with
trifloroacetic acid (TFA) (3 mL) with DCM as solvent or HCl
(1 mL) in methanol at room temperature overnight. The reac-
tion was then concentrated with a rotary evaporator. The solid
was dissolved in methanol, filtered, and precipitated in diethyl
ether. The solid was collected. Representative characterization
data follow. 1H NMR (CD3OD, δ, ppm): 1.0-1.2 (b (-CH3)),
2.4-2.65 (b (-CHCH2S-)), 2.65-3.1 (b (-CCHCH3CH2-));
(-SCH2CH-), 3.4-3.6 (b (-NCH2CH2-)), 3.61-3.78 (b
(-COOCH3)), 4.35-4.55 (b (-SCH2CH-)). 13C NMR (CD3-
OD, δ, ppm): 19 (-CH3), 35 (-CHCH2S-), 37 (-CHCH2S-),
38 (-SCH2CH-), 46 (-NCH2CH2-), 54 (-COOCH3), 55
(-SCH2CH-), 170 (-CHCOOCH3-), 178 (-NCOOCH-).
IR: 1640 (tertiary amide (C-N)), 1680 (ester (CdO)), 3350
(O-H, N-H), 3200 (O-H, N-H).
a Reagents and conditions: (i) HS-R, butylamine resin, tetrahydro-
furan (THF), room temperature; (ii) MeOTs, acetonitrile, 70 °C, 24 h.
The functionalized monomers were prepared by reaction with
the desired thiol and 2-isopropenyl-2-oxazoline to afford three
different functionalized 2-oxazoline monomers (Scheme 1:
monomers 1-3). While this reaction can be catalyzed by a
solution phase amine, an amino solid support was used to
simplify purification. This resin was prepared by reacting Merri-
field’s resin, an insoluble polymer support consisting of chlor-
omethylated polystyrene (1% cross-linked), with butylamine
(Scheme 2).36 The presence of the secondary amine on the solid
support was verified using IR spectroscopy. Though initially the
thiol-ene reactions were stirred overnight, later kinetic studies
show that the reactions were complete in less than 1 min. The
three functionalized monomers were then polymerized by catio-
nic ring-opening mechanism with initiation from methyl tosylate
followed by termination with water (Scheme 3) to yield three
functionalized polymers (Scheme 1: polymers 4-6).
Results and Discussion
To demonstrate the versatility of utilizing the thiol-ene to
prepare a range of functional oxazoline monomers, thiols of
varying reactivity were screened. Three representative thiols were
selected: thiophenol to first verify the coupling procedure, methyl
thioglycolate to investigate the compatibility of the polymeriza-
tion with hydrogen bond accepting functionalities, and N-(tert-
The thiol-ene coupling reactions were carried out in THF
at room temperature under nitrogen gas with a slight excess of
2-isopropenyl-2-oxazoline. The crude reaction mixtures were
purified solely by filtration to remove the solid support and
butoxycarbonyl)-L-cysteine methyl ester as a route toward in-
corporating peptides or proteins.

Article
Macromolecules, Vol. 43, No. 9, 2010 4085
concentration in vacuo to remove both the solvent and unreacted
2-isopropenyl-2-oxazoline.
monomers by the shift of the methylene protons of 2-isopropenyl-
2-oxazoline from singlets (Figure 1, b) at 5.4 and 5.7 ppm to
multiplets between the 2.7 and 2.9 ppm range for all three mono-
mers. Also, the singlet attributed to the methyl protons adjacent
to the double bonds (Figure 1, a) in 2-isopropenyl-2-oxazoline at
1.9 ppm is shifted to 1.2 ppm and is split to form a doublet. 13C
NMR (Figure S1) verified the shift of the terminal carbon of the
alkene from 122 ppm between 31 and 35 ppm for the corresponding
methylene adjacent to the thio ether. The disubstituted vinylic
carbon with a resonance of 133 ppm shifts between 34 and
37 ppm corresponding to a methyne carbon in the product. Finally,
the methyl at 19 ppm shifts to 17 ppm due to loss of the adjacent
double bond.
GC-MS spectroscopy (Figures S2 and S3) was used to validate
the purity of the 2-isopropenyl-2-oxazoline monomers. The gas
chromatograph showed a shift in retention time for the three
functionalized monomers with no contaminations by the starting
materials. The mass spectra exhibited the expected molecular ions
for all the monomers: 221.0 m/z (theoretical m/z: 221.3) for 1,
217.3 m/z (theoretical m/z: 217.3) for 2, and 346.5 m/z (theoretical
m/z: 346.4) for 3.
The functionalized monomers were characterized using 1H
(Figure 1) and 13C (Supporting Information Figure S1) nuclear
magnetic resonance (NMR) spectroscopy, and infrared (IR)
spectroscopy (Figure 2), and gas chromatography-mass spectro-
metry (GCMS) (Supporting Information Figures S2 and S3) to
confirm purity of the monomer and electrospray ionization (ESI)
mass spectroscopy (Supporting Information Figure S4) to pro-
1
vide higher resolution mass spectral data. H NMR (Figure 1)
indicated quantitative coupling in the synthesis of the three
Scheme 2. Synthesis of Secondary Amine Solid Support Catalyst in
Dimethylformamide (DMF), 70 °C
The use of ESI mass spectroscopy (Figure S4) allowed for
increased resolution of the mass spectra relative to the GC-MS to
provide further evidence for the quantitative thiol-ene coupling.
The ESI spectrum of 1 shows the monoisotopic molecular ion
peak at 244.078 m/z (theoretical m/z: 244.078) corresponding to
C12H15NOS þ Na and a monoisotopic peak at 222.0925 m/z
(theoretical m/z: 222.0945) corresponding to C12H15NOS þ H.
The spectra of 2 show the monoisotopic molecular ion peak at
240.070 m/z (theoretical m/z: 240.067) corresponding to
C9H15NO3S þ Na and a monoisotopic peak at 218.088 m/z
(theoretical m/z: 218.085) corresponding to C9H15NO3S þ H.
The spectra of 3 show the monoisotopic molecular ion peak at
369.152 m/z (theoretical m/z: 369.156) corresponding to
C15H26N2O5S þ Na and a monoisotopic peak at 347.170 m/z
(theoretical m/z: 347.164) corresponding to C15H26N2O5S þ H,
all in agreement with the predicted values.
Scheme 3. Polymerization Scheme of Functionalized 2-Isopropenyl-
2-oxazolinesa
The IR of 1 (Figure 2) shows the loss of the alkene absorbance
of the 2-isopropenyl-2-oxazoline starting material and new
absorbances at 3080 and 3050 cm-1 corresponding to the aromatic
a All polymers were purified by precipitation in diethyl ether.
Figure 1. 1H NMR spectra of functionalized 2-oxazoline monomers (CDCl3): (A) 2-isopropenyl-2-oxazoline; (B) monomer 1; (C) monomer 2; (D)
monomer 3.

4086 Macromolecules, Vol. 43, No. 9, 2010
Cortez and Grayson
Figure 2. IR spectra of functionalized 2-isopropenyl-2-oxazoline monomers: (A) 2-isopropenyl-2-oxazoline; (B) monomer 1; (C) monomer 2; (D)
monomer 3.
Figure 3. 1H NMR spectra of functionalized 2-isopropenyl-2-oxazoline polymers (CDCl3): (A) polymer 4; (B) polymer 5; (C) polymer 6.
C-H bond stretch, 1600 cm-1 corresponding to the aromatic
double bond stretches, and 1670 cm-1 corresponding to the imine
bond stretch. The IR of 2 shows new absorbances at 1740 cm-1
corresponding to the carbonyl bond stretch, 1250 cm-1 corre-
sponding to the ester O-C bond stretch, and 1670 cm-1
corresponding to the imine bond stretch. The IR of 3 shows
new absorbances at 1720 cm-1 corresponding to the carbonyl
bond stretch, 1250 cm-1 corresponding to the ester O-C bond
stretch, 1760 cm-1 corresponding to the acylic amine O-C bond
stretch, 3200 and 3350 cm-1 corresponding to the N-H bond
stretch, and 1670 cm-1 corresponding to the imine bond stretch.
Once the functionalized 2-isopropenyl-2-oxazoline monomers
were synthesized, they were then polymerized through a cationic
ring-opening mechanism by initiation from methyl tosylate
followed by termination with water (Scheme 3). The successful
polymerization of all three monomers and preservation of

Article
Macromolecules, Vol. 43, No. 9, 2010 4087
Figure 4. IR spectra of functionalized 2-isopropenyl-2-oxazoline polymers: (A) polymer 4; (B) polymer 5; (C) polymer 6.
Figure 6. Representative matrix-assisted laser desorption/ionization
time of flight (MALDI-TOF) mass spectroscopy for polymer 5.
MALDI distribution exhibits a repeat unit mass of 217.1 (theoretical
217.1) and an averageþresidual mass (when corrected for ionization by
complexation with Na ) of 105.0 m/z, which corresponds closely to the
expected chain transfer end-groups of -H and -SCH2COOCH3 with a
combined theoretical mass of 106.0 m/z.
resonances from 54 and 68 ppm corresponding to the two
adjacent methylene carbons in the 5-membered ring to one broad
signal at 45-48 ppm assigned to the methylene carbons of the
polymer backbone. There is also a slight shift in signal of the
tertiary carbon of the 5-membered ring from 170 to 176 ppm with
the formation of an amide carbonyl during the ring-opening.
IR was also used to verify the rather substantial functional
group changes during the polymerizations (Figure 4). The loss of
the imine absorbance at 1670 cm-1 and the appearance of the
tertiary amide stretching at 1640 cm-1 for all three polymers
verify the ring-opening polymerization. Also, the appearance of
peaks in all three polymers between 3300 and 3500 cm-1
corresponds to the hydroxyl terminus of the polymer and water
bound to the hydrophilic backbone. The IR also shows expected
absorbance of the side-chain functionalities for the phenyl
thioether 4 at 3080 and 3050 cm-1 corresponding to aromatic
C-H stretch and 1600 cm-1 corresponding to the aromatic
double bonds. For the methyl thioglycolate side chains of 5, an
Figure 5. Representative gel permeation chromatograms of functiona-
lized 2-isopropenyl-2-oxazoline polymers (THF as eluent, 2 mg/mL
sample in THF): (A) polymer 4; (B) polymer 5; (C) polymer 6.
1
side-chain functionalities were confirmed by H (Figure 3) and
13C NMR (Supporting Information Figure S5), IR (Figure 4), gel
permeation chromatography (GPC) (Figure 5, Table 1), and
matrix-assisted laser desorption/ionization time of flight
(MALDI-TOF) mass spectroscopy (Figure 6, Supporting Infor-
mation Figure S6, Table 1).
1H NMR (Figure 3) indicated polymerization by the broad-
ening of all the proton peaks of the three monomers combined
with the shift of the proton resonances between the 3.75-3.85
ppm range (Figure 1, d) and the 4.15-4.2 ppm range (Figure 1, e)
corresponding to methylene units in the 5-membered ring to one
broad peak spanning 3.1-3.6 ppm (Figure 3A, d) for 4, 3.4-3.6
ppm (Figure 3B, d) for 5, and 3.1-3.4 ppm (Figure 3C, d) for 6.
13C NMR (Figure S5) also showed a shift of the carbon
additional ester carbonyl absorbance was observed at 1735 cm-1
.

4088 Macromolecules, Vol. 43, No. 9, 2010
Cortez and Grayson
For the protected cysteine 6, a series of new absorbances were
observed at 1710 cm-1 corresponding to the carbonyl stretch,
1760 cm-1 corresponding to the acylic amine O-C stretch, and
3200 and 3350 cm-1 corresponding to the N-H stretch.
molecular weight range of 4000-4600, degrees of polymerization
from 16 to 23, and polydispersities below 1.4. However, a higher
molecular weight polymer sample (Mn = 9100) could be isolated
by fractionation of polymer 5f (detailed work-up in Experimental
Section). Finally, the sterically bulky N-(tert-butoxycarbonyl)-L-
Attempts to polymerize these functional oxazoline monomers
yielded consistent molecular weights with low degrees of poly-
merization (DP) and narrow polydispersities (PDI). Table 1 docu-
ments the number-average molecular weights (Mn), PDIs, and
DPs with varying reaction conditions for a series of polymeriza-
tions as reported by GPC (representative, Figure 5) for the three
polymers and MALDI-TOF MS (Figure S6 (4), Figure 6 (5)) for
the polymers from the benzyl 4 and methyl thioglycolate 5
functionalized. Though other groups have shown that polymers
of 2-oxazolines can be polymerized with molecular weights of up
to 15 000 with the use of a microwave reactor37 and without
microwave catalysis for the case 2-ethyl-2-oxazoline,38 attempts
to prepare thioether-functionalized polymers without microwave
catalysis yielded polymers with more modest molecular weights.
The polymerization of the phenylsulfide-functionalized mono-
mer, 1, resulted in polymers 4a-e, which exhibited low molecular
weights with a range between 3800 and 4300, degrees of polym-
erization from 16 to 19, and polydispersities below 1.3. The acid-
catalyzed polymerization of the methyl thioglycolate-functiona-
lized monomer, 2, resulted in polymers 5a-e with a similar
cysteine methyl ester-functionalized monomer, 3, yielded poly-
mers 6a-d which again exhibited a modest molecular weight
range from 2800 to 5000, degrees of polymerization between 8
and 14, and polydispersities below 1.4. For all three monomers,
all attempts failed to prepare higher molecular weight polymers
despite variations in the reaction conditions, suggesting the
possibility of chain transfer or other side reactions. Recently,
Kempe et al.39 proposed that the cationic ring-opening poly-
merization of a 2-oxazoline monomer containing a thioether
bond was not living due to the nucleophilic character of that
functionality. The thioether was proposed to carry out nucleo-
philic attack on the propagating oxazolium species, generating a
sulfonium ion, followed by a chain transfer event. The possibility
of chain transfer was supported by MALDI-TOF MS end-group
analysis which in the case of polymer 5e afforded a combined
residual mass of 105 Da, close to the expected 106 Da for
initiation from Hþ and termination with thioglycolate. In addi-
tion, a much weaker signal was observed which corresponded to
the original initiation from the methyl cation and termination
with thiophenol for polymer 4 (Figure S6). Kinetic studies which
compared the conversion of monomer 1 to the molecular weight
of polymer 4 exhibited a molecular weight that sharply increased
at low monomer conversion and then plateaued despite contin-
ued conversion of monomer, providing further evidence of the
nonliving character of the polymerization (Figure 7).
The successful prepolymerization introduction of functionality
via thiol-ene coupling was confirmed for a diversity of func-
tional monomers. However, because of the incompatibility of
many basic or nucleophilic functionalities with the cationic
polymerization conditions, protecting groups had to be used
with side chains including both amines and carboxylic acids. The
deprotection reactions of 5 to remove the methyl ester as well as
the removal of the amine and carboxylic acid functionalities of 6
were next explored and characterized. The deprotection of the
methyl ester of 5 was verified by a loss of the methyl ester
Figure 7. Number-average molecular weight (Mn) and conversion plot
for the polymerization of 4 in acetonitrile at 65 °C.
1
resonance at 3.4-3.6 ppm in the H NMR (Figure 8, f) and at
Figure 8. 1H NMR spectra of (A) polymer 5 (CDCl3) and (B) deprotected thioglycolate-functionalized 2-isopropenyl-2-oxazoline polymer (D2O).
The absence of the methyl ester protons (f) suggests the deprotection occurred.

Article
Macromolecules, Vol. 43, No. 9, 2010 4089
Figure 9. 1H NMR spectra of (A) polymer 6 (DMSO) and (B) deprotected
L-cysteine methyl ester-functionalized 2-isopropenyl-2-oxazoline polymer
(CD3OD). The absence of the methyl tert-butyl protons (h) indicates the deprotection occurred. (C) Deprotected N-(tert-butoxycarbonyl)-
L-cysteine-
functionalized 2-isopropenyl-2-oxazoline polymer (DMSO). The absence of the methyl ester protons (g) indicates the deprotection occurred.
51 ppm in the 13C NMR (Supporting Information, Figure S7, f)
as well as a shift in the IR (Supporting Information, Figure S8)
protection group to expose an N-terminus on each repeat unit or
the methyl ester protection group to expose a carboxylic acid on
each repeat unit was demonstrated, highlighting the compatibil-
ity of this coupling and polymerization chemistry with conjuga-
tion to peptides and proteins. The diversity of side-chain
modifications combined with the water solubility of biocompat-
ibility of the poly(oxazoline) backbone make this synthetic route
an attractive method for preparing polymer bioconjugates.
Because the attachment occurs through the side chains rather
than the end groups of the polymer, a much higher degree of
modification can be obtained than with traditional poly(ethylene
glycol) end-group conjugation.
from 1735 cm-1 for the carbonyl stretching to 1660 cm-1
.
After the deprotection of the tert-butoxycarbonyl group on 6,
a loss of the tert-butyl protons at 1.25-1.5 ppm in the 1H NMR
(Figure 9A,B, h) and the carbon resonances at 29 ppm in the 13
C
NMR (Supporting Information, Figure S9A,B, i) was observed.
The loss of the tertiary carbon resonance at 81 ppm (Figure S9A,
B, j) and the carbonyl carbon resonance at 156 ppm is also
observed (Figure S9A,B, k). The loss of the acylic amine O-C
bond stretch at 1760 cm-1 observed in the IR (Supporting
Information, Figure S10) further suggests the deprotection of
the tert-butyl group. Characterization of the methyl ester depro-
tection of 6 shows an absence of the methyl ester protons at
3.61-3.78 ppm in the 1H NMR (Figure 9A,C, g) and at 52 ppm in
the 13C NMR (Figure S9A,C, f). The IR shows a shift from
1710 cm-1 corresponding to the carbonyl bond stretch to
1680 cm-1 (Figure S10). After quantitative deprotection of the
methyl ester functionality of 5 and the amine and the carboxylic
acid functionalities of 6, these diverse polymers now have the
potential for use in biological applications, including the further
reaction of the cysteine functionalized polymer 6 to yield peptide
or protein conjugates.
Acknowledgment. The authors thank the Tulane University
for support of this research and the Louisiana Board of Regents
for a graduate fellowship (MAC). The authors also acknowledge
the National Science Foundation (NSF-MRI 0619770) for
enabling mass spectral characterization.
Supporting Information Available: ESI spectra of mono-
mers, GCMS of starting materials and monomers, 13C NMR of
all monomers and polymers, representative MALDI-TOF spec-
trum of polymer 4, and IR of the deprotection reactions of
polymers 5 and 6. This material is available free of charge via the
Internet at http://pubs.acs.org.
Conclusions
The prepolymerization functionalization of oxazoline mono-
mers has been investigated for the installation of diverse func-
tionality along the poly(N-acylethylenamine) backbone.
Through the use of thiolene coupling between a thiol and the
pendant alkene of 2-isopropenyl-2-oxazoline, the monomer
functionalization route has enabled the preparation of PNAI
with aryl, ester, amine, and carboxylic acid side chains. The
resulting polymers could be prepared with molecular weights
ranging from 3000 to 8000 and polydispersities typically below
1.3. Of particular interest, a protected cysteine amino acid was
successful coupled to the 2-isopropenyl-2-oxazoline monomer
and polymerized. The selective deprotection of either the t-BOC
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