MALDI-TOF MS investigation of the unconventional termination of living polyoxazoline with ammonia

Article abstract of DOI:10.1002/pola.28495

Poly(2-oxazoline) (POx) based materials have experienced renewed interest, due to their biocompatibility and broad functional diversity. Although research pertaining to cationic ring opening polymerization (CROP) of 2-oxazoline derivatives spans upwards of 5 decades, modern characterization techniques, specifically Matrix Assisted Laser-Desorption Ionization—Time of Flight Mass Spectrometry (MALDI-TOF MS), were not available during early studies to aid in identifying polymer end-groups. Through careful synthesis and analytical characterization, evidence supports an alternative mode of nucleophilic attack during ammonia termination, with nucleophilic attack occurring at a different site on the terminal oxazolinium than where polymer propagation occurs. Furthermore, investigations employing targeted end-group modification, in conjunction with MALDI and NMR analysis, determined the structure of the resultant terminal group was a hydroxyethylamino end group. Based on the observed data, a mechanistic explanation for the observed 2-oxazoline ring-opening termination is proposed.

Full text of DOI:10.1002/pola.28495

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MALDI-TOF MS Investigation of the Unconventional Termination of
Living Polyoxazoline with Ammonia
Karolina A. Kosakowska,1 Philip Dimitrov,2 Gangadhar Panambur,2 Scott M. Grayson1
1Department of Chemistry, Tulane University, New Orleans, Louisiana, 70118
2Materials Science Research and Development, MilliporeSigma, Milwaukee, Wisconsin, 53209
Correspondence to: S.M. Grayson (E-mail: sgrayson@tulane.edu)
Received 19 October 2016; accepted 24 December 2016; published online 00 Month 2017
DOI: 10.1002/pola.28495
ABSTRACT: Poly(2-oxazoline) (POx) based materials have expe-
rienced renewed interest, due to their biocompatibility and
broad functional diversity. Although research pertaining to cat-
ionic ring opening polymerization (CROP) of 2-oxazoline deriv-
atives spans upwards of 5 decades, modern characterization
techniques, specifically Matrix Assisted Laser-Desorption Ioni-
zation—Time of Flight Mass Spectrometry (MALDI-TOF MS),
were not available during early studies to aid in identifying
polymer end-groups. Through careful synthesis and analytical
characterization, evidence supports an alternative mode of
nucleophilic attack during ammonia termination, with nucleo-
oxazolinium than where polymer propagation occurs. Further-
more, investigations employing targeted end-group modifica-
tion, in conjunction with MALDI and NMR analysis, determined
the structure of the resultant terminal group was a hydroxye-
thylamino end group. Based on the observed data, a mechanis-
tic explanation for the observed 2-oxazoline ring-opening
C
V
termination is proposed.
2017 Wiley Periodicals, Inc. J.
Polym. Sci., Part A: Polym. Chem. 2017, 00, 000–000
KEYWORDS: ammonia termination; end group analysis; end
group modification; MALDI-TOF MS; polyoxazoline
philic attack occurring at
a different site on the terminal
high-purity reagents and anhydrous conditions to mitigate
premature termination by nucleophiles or chain-transfer
reactions.39–42 Furthermore, such side reactions become
increasingly likely at higher degrees of polymerization.43–45
Nonetheless, the scalable synthesis of end-functional POx
derivatives suitable for bioconjugation has been extensively
reported, such as alkyne-/azide-functionalized,46–49 and
thio-/alkene-functionalized50–52 derivatives suitable for Huis-
gen53 and hydrothiolation54 “click” reactions, respectively, as
well as carboxyl55–57 and amine24,58–60 moieties for alterna-
tive conjugation reactions. In most cases, a functional initiat-
ing and/or terminating reagent is employed during
INTRODUCTION Polyoxazolines (POx) are among the most
tunable family of polymers, with access to readily derivatized
2-, 4-, and 5-substituted monomers allowing an exceptionally
broad range of target applications.1–5 In addition, functionali-
zation of pendant groups pre- or post-polymerization pro-
vide a route to further diversity.6–9 This tunable nature of
POx has made them an attractive target for application-
driven investigations,10–13 particularly in the case of bio-
medically relevant materials, due to their pseudo-peptide
backbone structure,14–16 adjustable thermoresponsive behav-
ior,17–19 tailorable solubility,20,21 biocompatibility, and in vivo
clearance properties.22–27 Unlike poly(ethylene glycol), the
current life-sciences workhorse for water-soluble bioconju-
gates,28–30 POx can incorporate addressable functionalities
on their side chains (in addition to their end groups) for bio-
materials applications.31–34 This exceptional versatility has
encouraged research into new monomers, optimized poly-
merizations, and novel applications.
polymerization, to afford end-functional material via
straightforward “one pot” polymerization strategy.61
a
However, amine end-functionalized POx remains difficult to
prepare due to synthetic challenges. Protected amine initia-
tors are prohibitive, as most exhibit a nucleophilic lone pair,
which can terminate the chain, and extractable protons,
which can lead to chain transfer reactions.62,63 Conversely,
most strategies introducing an amine group via termination
are ill-suited for meeting production scale demands, requir-
ing an additional post-termination reaction. For example,
Tauhardt et al. obtained POx with a primary amine terminal
However, the large-scale manufacturing of well-defined, mul-
tifunctional POx materials remains a challenging endeavor.
Oxazoline polymers are typically synthesized via cationic
ring-opening polymerization (CROP),35–38 which requires
Additional Supporting Information may be found in the online version of this article.
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TABLE 1 Obtained PEtOx Molecular Weight Properties via SEC, Titration, and MALDI
Target (Theoretical) SEC
Titration
MALDI-TOF MS
-
D
-
D
Sample
No. RU
Mn
Mn
Mn
Mn
PEtOx20
PEtOx50
PEtOx100
20
2000
1900
4500
9400
1.14
1.12
1.12
2000
5500
8000
1830
1.03
1.02
1.01
50
5000
4970
100
10,000
10,260
group via direct potassium phthalimide end-capping, but
required subsequent hydrazine treatment of the phthalimide
adducts.64,65 Similarly, Luxenhoffer et al. report a two-step
amino-termination involving N-Boc-piperazine termination,
followed by acid-catalyzed protecting group removal, to
afford POx with a secondary amine end group.66,67 The only
“one pot” procedure reported to yield primary amine func-
tional POx was introduced by Kobayashi et al. nearly three
decades ago, and involved the single-step CROP termination
with ammonia.68 This simple synthetic strategy appears to
be the most attractive pathway for large-scale POx synthesis,
and therefore, was explored in detail with modern character-
ization techniques.
America, Portland, OR). Acetic anhydride (ꢀ97%, Cat. # A10)
and tetrahydrofuran (THF) (Cat. # T397) were purchased
from Fisher Scientific (Thermo Fisher Scientific, Waltham,
MA). THF was distilled from CaH before use.
Characterization
Size exclusion chromatography (SEC) was performed at 25
8C with a Waters Alliance e2695 module, a Waters 2414 RI
detector and a series of three Waters Styragel HR 1, HR 2 (5
lm, 300 3 7.8 mm2) and HR 3 (3 lm, 150 3 6 mm2) col-
umns (Waters Corporation, Milford, MA). THF with 5 vol %
of triethylamine was used as an eluent, and a molecular
weight calibration curve was applied based on narrow dis-
persity polystyrene standards.
The current contribution focuses on applying this ammonia-
termination procedure to the large-scale synthesis of well-
defined amino-terminated POxs. Poly(2-ethyl-2-oxazoline)s
(PEtOx)s of varying degrees of polymerization were prepared
via living cationic polymerization of 2-ethyl-2-oxazoline, fol-
lowed by quantitative termination with a methanolic solution
of ammonia. However, instead of yielding the targeted prima-
ry amine end group, data suggested that an alternative
mechanism of POx termination took place. Comprehensive
material characterization of the alternative ammonia-
terminated products was carried out through titration,
MALDI-TOF MS, and NMR spectroscopy with targeted end
group modification employed to confirm end group struc-
ture.69 Additionally, a parallel investigation was conducted
with short PEtOx oligomers to elucidate the end group struc-
ture and small-molecule byproducts formed during the ter-
mination of PEtOx with ammonia.
Mass spectral data were obtained using a Bruker Autoflex III
MALDI-TOF MS (Bruker Daltonics, Billerica, MA). Samples
were acquired via positive-ion reflector mode with a 20 kV
acceleration voltage and delayed extraction, using Bruker Dal-
tonics flexControl 3.0 software. All mass measurements were
calibrated using SpheriCal dendritic calibration standards
(Polymer Factory, Stockholm, Sweden). Matrix and counterion
solutions of 1,8,9-trihydroxyanthracene (dithranol, 97%, Alfa
Aesar) and sodium trifluoroacetate (NaTFA, 98%, Aldrich)
were prepared in THF at 20 and 2 mg/mL, respectively, and
mixed with 2 mg/mL analyte in THF at a 3:1:3 matrix:counter-
ion:analyte ratio. Samples (3 lL) were applied to the target via
the dried drop method and data collected using the minimum
laser power that exhibited a significant signal-to-noise ratio.
Mass assignment and analysis was performed on Bruker Dal-
tonics FlexAnalysis 3.0 software.
NMR analysis was performed on a Varian system (Agilent
Technologies, Santa Clara, CA) operating at 500 MHz using
methanol-d4 as solvent at room temperature. Spectral analy-
sis was performed using Mestrenova 9.0.1 software (Mestre-
lab Research SL, Santiago de Compostela, Spain).
EXPERIMENTAL
Materials
Reagents methyl trifluoromethanesulfonate (ꢀ98%, Cat.#
164283), barium oxide (BaO) (99.99%, Cat.# 554847), anhy-
drous chlorobenzene (99.8%, Cat.# 284513), and 7M ammonia
in methanol (Cat.# 499145) were obtained from Sigma-
Aldrich (Sigma-Aldrich, St. Louis, MO) and used as received. 2-
ethyl-2-oxazoline (>99%, Cat.# 137456) was stirred over BaO
for 72h and distilled under reduced pressure before use. End
group modifying reagents sodium hydride (NaH) (95%, Cat.#
223441), calcium hydride (CaH) (95%, Cat. #208027), trime-
thylamine (>99%, Cat.# T0886), benzyl bromide (98%, Cat.
#B17905), allyl bromide (97%, Cat. #337528), and thiophos-
gene (Cat.# 115150) were used as received from Sigma-
Titrations were performed against 0.1 M HCl aqueous solu-
tion on a Metrohm 835 Titrando automatic titrimeter (Met-
rohm AG, Herisau, Switzerland). Measurements were run
under ambient conditions.
Polymerization and Ammonia-Termination of
Functional PEtOx
The polymerization of 2-ethyl-2-oxazoline was performed in
an air and moisture free environment similar to the proce-
dure described in Ref 68. Predetermined amounts of mono-
mer, methyl trifluoromethanesulfonate (MeOTf) initiator and
Aldrich. 2,4-Dinitrofluorobenzene (Cat.
obtained from TCI America (Tokyo Chemical Industry Co.
# 70-34-8) was
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chlorobenzene solvent were mixed together in 40 mL culture
tubes in a glove box. Polymerizations were performed at 70
8C for 24 h to ensure a complete monomer conversion.
Ammonia (7 M) in methanol was added to the living poly-
mer (203 molar excess of ammonia with respect to the poly-
mer’s terminal group) at 0 8C and stirred for 2 h. The
temperature was then increased to 25 8C and the reaction
was allowed to continue for 24 h at this temperature. Sol-
vents were removed under reduced pressure, and polymer
residue dissolved in DI water and dialyzed against DI water
for 24 h before the product was recovered by freeze drying
in 85 wt% yield.
End Group Modification of Ammonia-Terminated PEtOx
Representative 2,4-Dinitrofluorobenzene Substitution
Reaction
Ammonia-terminated PEtOx20 (60 mg, 0.03 mmol) was dis-
solved in 3 mL distilled THF, followed by addition of 50 lL
(0.4 mmol) of 1-fluoro-2,4-dinitrobenzene while stirring. The
reaction was monitored for color change (less than 1 min)
indicating completion. The product was analyzed without
additional workup.
Representative Alkylation Reaction
Ammonia-terminated PEtOx20 (40 mg, 0.02 mmol) was dis-
solved in distilled THF (5 mL) under inert N2 atmosphere.
95% NaH (10 mg, 0.4 mmol) was added to the reaction ves-
sel and stirred for 10 min prior to addition of 95 lL (0.8
mmol) benzyl bromide. The system was stirred at ambient
temperature (typically, 2–6 days) and the reaction monitored
by MALDI-TOF MS characterization of reaction aliquots until
comprehensive end group transformation was observed.
Residual NaH was quenched with cold MeOH and the mixed
solvents were removed under reduced pressure. Residue
was dissolved in 5 mL DCM, washed with H2O and brine,
and dried over anhydrous Na2SO4. The solution was concen-
trated prior to product precipitation into cold DEE and vacu-
um drying.
Representative Thiophosgene Coupling Reaction
Ammonia terminated PEtOx20 (13 mg, 0.006 mmol) was dis-
solved in 2 mL distilled THF under inert N2 atmosphere, fol-
lowed by addition of 3 lL trimethylamine (2.2 mg, 0.022
mmol) while stirring. Thiophosgene (0.9 mg, 0.007 mmol)
was added and the reaction was allowed to proceed for 5
min prior to quenching with saturated aq. NaHSO4. The
product was extracted from the reaction mixture 3x using
DCM and the combined organic layers were dried over anhy-
drous Na2SO4 prior to solvent removal.
FIGURE 1 Comparison of MALDI-TOF mass spectra obtained of
PEtOx20 (Mn 5 2000) for (a) K1 ionization; (b) Na1 ionization; (c)
Li1 ionization; trace K1 ionized species seen in spectra (c).
Obtained mass values agree with hyrdoxyethylamino end group
assignment, with no primary amine end group observed. [Color
figure can be viewed at wileyonlinelibrary.com]
RESULTS AND DISCUSSION
Because purified monomer and exhaustive anhydrous, air-
free conditions were used, the cationic ring opening poly-
merization (CROP) of 2-oxazolines should be able to achieve
a “living polymerization” particularly at lower molecular
weights. All polymerizations were allowed to proceed to
complete monomer conversion prior to termination with
ammonia. The molecular weight characteristics of the
polymers are tabulated in Table 1. The target number aver-
age molecular weight (Mn) of the obtained PEtOx correlates
closely with the theoretical Mn values as revealed by SEC,
MALDI and titration measurements, providing evidence for
the living nature of the polymerization. In addition, SEC
curves of all of the samples exhibit narrow and symmetrical
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TABLE 2 Overview of End Group Modifying Reagents
confirming, as described by Kobayashi et al., that the majori-
ty of the polymer chains bear a single amino group. MALDI-
TOF mass spectral distributional analysis yields Mn values
that are in close agreement with both SEC and titration find-
Monoisotopic DMn
Reagent
Structure
Per Unit Added (Da)
-
ings. Although the MALDI-TOF MS calculations of D are much
Allyl Bromide
40.03
42.01
lower than observed by SEC, this deviation is typical when
comparing these techniques.
Acetic Anhydride
Benzyl Bromide
90.05
MALDI-TOF MS Analysis via Select End Group
Modification
1-Fluoro-2,4-Dinitrobenzene
166.00
In the three decades since Kobayashi et al. reported their
termination of POx with ammonia, MALDI-TOF mass spec-
trometry has evolved as a tool to provide much more
detailed end group characterization. Unlike traditional char-
acterization techniques (i.e., NMR, FTIR) where the signals
for the end groups become vanishingly small with increasing
molecular weight, the amplitude of the end group signal in
MALDI-TOF MS does not diminish with increasing molecular
weight. As a results, MALDI analysis enables precise calcula-
tion of the mass of the repeat units and end groups, as well
as distributional properties, such as the Mn and the
dispersity.70
Thiophosgene
41.96
distributions (Figs. S1–S3, Supporting Information), with dis-
-
persity (D) values less than 1.15, also consistent with a liv-
ing polymerization process (Table 1). It was necessary to use
a THF/TEA eluent system for SEC analyses, to mitigate inter-
actions between each polymer’s amino end group and the
SEC column which causes an observed tailing in the chroma-
tograms. Values of Mn obtained by titration of the amino end
group with HCl were close to the theoretical values,
FIGURE 2 Comparison MALDI-TOF mass spectra obtained from end group modification of PEtOx20 with Na1 ionization, monitor-
ing 16-mer peak shift; (a) unmodified PEtOx20; (b) allyl bromide conjugate; (c) acetic anhydride conjugate; (d) benzyl bromide con-
jugate; (e) 2,4-dinitrofluorobenzene conjugate; (f) thiophosgene conjugate. [Color figure can be viewed at wileyonlinelibrary.com]
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Utilizing an 8-point dendrimer calibration for precise mass
calculation, end group characterization was performed via
MALDI-TOF MS analysis of the ammonia-terminated PEtOx
species. In order to confirm the mode of ionization, the
PEtOx samples were analyzed using 3 different cations, K1,
Na1, and Li1, as the ionizing species. As seen in Figure 1,
the observed mass spectra for all PEtOX20 samples exhibit a
narrow polymer distribution with neighboring peak masses
differing by 99.07 Da, corresponding to the mass of a 2-ethyl-
2-oxazoline repeat unit. The 16 mass unit difference between
the spectra ionized with K1 versus Na1, and Na1 versus Li1,
confirm that each spectrum corresponds to complexation with
a single alkali cation. Equivalent spectra for PEtOx50 and
PEtOx100 can be found in the Supporting Information (Figs. S4
and S5). Assignments for the potassiated mass spectrum are
provided as a representative interpretation [Fig. 1(a)]. The
major observed distribution, for example the peak at m/
z 5 1699.046, does not correspond to the methyl initiated,
amino terminated PEtOx 16-mer [CH3-PEtOx16-NH2 1 K]1 as
predicted by Kobayashi et al., which should instead be
observed at m/z 5 1655.101. On the other hand, this observed
signal is consistent with a more complicated termination
mechanism, which would generate a hydroxyethylamino end
group [CH3-PEtOx16-NHCH2CH2OH 1 K]1 with a theoretical
mass of 1699.127. Furthermore, the mass spectra show no
measurable signal of the a-methyl-x-amino PEtOX 16-mer, or
any alternative termination products for all three cations
examined. Only a trace amount of the potassiated polymer is
observed in the spectra of the lithiated sample, a consequence
of residual salts in glassware. Therefore, it can be concluded
that the exclusive product from ammonia termination of living
PEtOX is the hydroxyethylamino end group, and not the previ-
ously reported primary amine functionality.
To confirm this conclusion, all PEtOx species were subject to
targeted end group modification employing reagents of
known reactivity.71 The subsequent mass shifts observed
after these modifications could be used to verify the end
group assignments. A summary of reagents and correspond-
ing monoisotopic masses added per substitution can be
found in Table 2. Generally, reactions were conducted under
basic conditions using abundant excess of electrophile to
ensure the quantitative transformation of the terminal end
group. Allyl bromide (AB), acetic anhydride (AA), and benzyl
bromide (BB) were selected because they should react with
both the amino and hydroxyl functional groups on terminal
chain end. To compliment these reactions, 1-fluoro-2,4-
dinitrobenzene (DNFB), also referred to as Sanger’s reagent,
was also explored because displacement of its fluorine
occurs exclusively with primary and secondary amines.72
Finally, the reaction with thiophosgene (TPG) was explored
to confirm the proximity of the amino and alcohol function-
alities, as the proposed vicinal nature of the hydroxylethyla-
mino end group should enable the rapid formation of a 5-
membered oxazolidinethione ring.
FIGURE 3 Comparison of MALDI-TOF mass spectra obtained
for (a) K1 ionization; (b) Na1 ionization; (c) Li1 ionization of
ambient hydroxyl terminated PEtOx5 oligomer (Mn 5 600);
dashed box corresponds to hydroxyl end group; solid distribu-
tion corresponds to hydroxyethylamino end group formation
via transient atmospheric water termination. [Color figure can
be viewed at wileyonlinelibrary.com]
reaction, a comprehensive shift of the parent PEtOx20 distri-
bution was observed, indicating complete end group trans-
formation. Furthermore, the observed MS signals are in good
agreement with the expected post-modification end group
MALDI-TOF MS analysis results of PEtOx20 end group modifi-
cation are summarized in Figure 2. Following each coupling
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FIGURE 4 1H NMR spectra of PEtOx oligomer terminated with ammonia, (a) 1D and 2D NMR (CD3OD) of PEtOx5 oligomer; (b) 1D
and 2D NMR (CDCl3) of trifluoroacetylation reaction product, observed shifts of the h/i signals further confirm the proposed end
groups structure. [Color figure can be viewed at wileyonlinelibrary.com]
weights. AA is expected to react with all amines or alcohols
once. AB and BB, on the other hand will react with alcohols
once, secondary amines once, but primary amines twice. In
the case of AB, AA, and BB modifications, the addition of two
equivalents of reagent were observed, corresponding to a
single reaction with the terminal alcohol and an additional
reaction with the secondary amine functionalities of the
hydroxyethylamino terminus. Specifically considering the
equivalents of TPG should be consumed. However, the
observed cyclization would only occur if these functionalities
were within sufficient proximity to form a 5- or 6-membered
ring.
In summation, the combination of targeted end group modifi-
cations and MALDI-TOF MS analysis provide unambiguous evi-
dence for the generation of a hydroxyethylamino end group as
a result of ammonia termination during PEtOx CROP.
PEtOx20 16-mer ([MTheo 1 Na1] 5 1683.153 Da),
a post-
modification AB mass shift of 80.141 Da ([DMTheo] 5 80.063
Da) was observed, as well as an AA mass shift of 84.134 Da
([DMTheo] 5 84.021 Da), and BB mass shift of 180.141 Da
([DMTheo] 5 180.094 Da). Furthermore, a single DNB addition
to the secondary amine on CH3-(EtOx)16-NHCH2CH2OH
([DMTheo] 5 166.001 Da) generated a corresponding 166.286
Da mass shift. Because DNB only reacts with amines, this
single addition to the secondary amine, combined with the
previous amine and hydroxyl functionalizations, confirms the
alternative termination product. Lastly, the reaction with
TPG generates a 41.974 Da mass shift for the PEtOX 16-mer
([DMTheo] 5 41.956 Da), correlating to terminal cyclic thio-
carbamate ring formation. This result confirms the relative
position of the hydroxyl and secondary amino groups. If, for
example, the secondary amine and alcohol were both
present, but located some distance from each other, two
Analysis of Oligomeric PEtOx
In order to further confirm the identity and clarify the origin
of the hydroxyethylamino end group, an oligomeric PEtOx
was synthesized using only 5 equivalents of monomer per
initiator. Following polymerization, the CROP of the PEtOx
short-chain was subject to two parallel termination proce-
dures: active termination with excess methanolic ammonia
and passive “ambient” termination via exposure to atmo-
spheric water. The PEtOx oligomers were subjected to
detailed analysis to elucidate both the end groups and the
byproducts formed during CROP ammonia termination.
MALDI-TOF MS Analysis of PEtOx Oligomer Terminated
under Ambient Conditions
To better understand competing reactions during the ammo-
nia termination of PEtOx, a sample of oligomer that was
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passively terminated with adventitious water was analyzed
by MALDI-TOF MS. Unlike the ammonia end capping reac-
tions—which were kept under strictly anhydrous condi-
tions—the living propagating cation was quenched by
atmospheric water upon removal for analysis. As seen in Fig-
ure 3, overlapping distributions in the PEtOx5 MALDI spectra
indicate formation of two distinct end group species, corre-
sponding to classical hydroxyl termination and hydroxyethy-
lamino termination products.
Under the passive termination with water, oxazolinium ring
opening appears to yield both the traditional hydroxyl end
group and the alternative hydroxyethylamino end group.
Once again, MALDI-TOF MS was employed (Fig. 3) comparing
the addition of three different cations (K1, Na1 and Li1) to
the water-terminated PEtOx 7-mer. For each cation used, two
major distributions can be seen in the mass spectra: one cor-
responding to the alkali complex of the hydroxyl terminated
species (dashed box) and the other corresponding to the
alkali complex of the hydroxyethylamino terminated species
(solid box). Focusing on the potassiated species [Fig. 3(a)],
the peaks at m/z 5 807.354 and at m/z 5 764.592, corre-
sponding to [CH3-PEtOx7-NHCH2CH2OH 1 K]1 and [CH3-
PEtOx7-OH 1 K]1, respectively, are in good agreement with
theoretical values. Furthermore, each distribution shifts rela-
tive to the cation added, with a third minor distribution
which remains unchanged for all three spectra correspond-
ing to protonation of the amine on the hydroxyethylamino
termination product.
To confirm the identity of the end groups, these PEtOx
oligomers were subjected to the same end group modifica-
tion conditions and analysis as described above for the
ammonia terminated 20-mers. Results detailed in Figure S6
(Supporting Information) show the hydroxyaminoethyl distri-
bution (Fig. S6, Supporting Information, blue) shifts are con-
sistent with containing one amino and one hydroxyl group
within the terminal end group. The hydroxyl terminated dis-
tribution (Fig. S6, Supporting Information, green), on the oth-
er hand, exhibits a distinctly different set of shifts consistent
with a single hydroxyl terminal group: namely, single alkyla-
tions and single esterifications, and no reaction with DNFB.
FIGURE 5 1H NMR analysis of 1:1 monomer:initiator adduct (a)
reaction intermediate of “living” monomer directly after initia-
tion; (b) crude product following excess ammonia termination.
[Color figure can be viewed at wileyonlinelibrary.com]
propionitrile byproduct also exhibit a COSY correlation. Full
1H NMR spectra can be seen in the Supporting Information
(Figs. S7 and S8). These results further corroborate the con-
clusions from the thiophosgene modification, confirming the
vicinal nature of the primary hydroxyl group, and the sec-
ondary amine.
NMR Analysis of Ammonia Terminated PEtOx Oligomer
Model with Subsequent End Group Modification
The hydroxyethylamino end group was exclusively observed
in the 1H NMR spectrum of ammonia terminated oligomeric
PEtOx derivatives [Fig. 4(a)]. Integration for the resolved ter-
minal resonance i (3.62 ppm) and initiating methyl signal a
(3.02 ppm) relative to e, the methyl of the repeat unit (1.06
ppm) yielded a degree of polymerization of approximately 4,
close to the targeted value of 5. Homonuclear correlated
spectroscopy (COSY) NMR analysis [Fig. 4(c)] of the reaction
product displays signal correlation between the methylene
signals g (2.70 ppm) and f (3.54 ppm) on the penultimate
ethylene unit, as well as between the methylene signals h
(2.76 ppm) and i (3.62 ppm) on the terminal ethylene unit.
In addition, signals j (2.34 ppm) and k (1.24 ppm) of the
Furthermore, 1H NMR monitored end group modification
was performed on oligomeric PEtOx utilizing trifluoroacetic
anhydride (TFAA) [Fig. 4(b)]. Substitution occurring at the
secondary amine functionality resulted in a downfield shift
of adjacent methylene signals g and h, from 2.70 ppm and
2.77 ppm, respectively, in the unmodified polymer [Fig. 4(a)]
to 3.66 ppm and 3.88 ppm, respectively, in the TFAA coupled
product [Fig. 4(b)]. Similarly, the proton signal adjacent to
the terminal hydroxyl unit can be seen at 3.62 ppm for the
PEtOx oligomer [Fig. 4(a)], while after esterification the
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SCHEME 1 Proposed mechanism of POx ammonia termination (a) traditional termination mechanism via C2 attack for formation
of primary amine end group (not observed) (b) proposed ring opening termination mechanism via C5 attack to yield the observed
hydroxyethylamino end group.
deshielding causes a shift to 4.56 ppm [Fig. 4(b)]. COSY anal-
ysis of the TFAA modified [Fig. 4(c)] polymer also verifies
that the methylene adjacent to the ester (i, 4.56 ppm) is
attached to the methylene adjacent to the trifluoroacetylated
amino group (h, 3.88 ppm) further confirming the proximity
of the hydroxyl and amino groups on the N-(hydroxyethyl)a-
mino end group.
Thus, the two remaining peaks, located at 2.47 ppm and
1.30 ppm, can be assigned to the byproduct of PEtOx ammo-
nia termination from expulsion of the propyl fragment. These
observed resonances and splitting patterns are consistent
with the expected shifts for propionitrile, the expected
byproduct in the proposed mechanism below. Additional
structural confirmation via 13C NMR and GC MS can be
found in the Supporting Information (Figs. S9–S11); these
findings are in agreement with previous characterization
data, providing additional evidence to indicate nucleophilic
termination occurs at the C2 position, rather than the C5
position of 2-oxazoline.
NMR Analysis and Byproduct Elucidation via 1:1
Monomer:Initiator Reaction Terminated with Excess
Ammonia
To simplify byproduct identification, an activated (2-ethyl-2-
oxazoline) “unimer” was prepared and analyzed via NMR
analysis. Here, the unimer sample was prepared using identi-
cal PEtOx CROP conditions, but with equimolar amounts of
monomer and initiator. Immediately following MeOTf addi-
tion to the monomer, 1H NMR of the reaction mixture in
methanol-d4 [Fig. 5(a)] indicates formation of a “living unim-
er,” as expected in the case of CROP. Observed spectral split-
ting, chemical shifts and integrations of the intermediate are
in good agreement with ion pair assignment, supporting the
living nature of cationic 2-ethyl-2-oxazoline monomer.
Proposed Mechanism
Initially, it merits mentioning the driving force behind oxazo-
line ring-opening polymerization: the scission of the CAO
sigma bond in the relatively stable five-membered ring is off-
set by the isomerization to the thermodynamically favorable
amide.73 Therefore, CROP chain growth proceeds via mono-
mer attack at the C5 oxazolinium position and subsequent
CAO bond scission. Nucleophilic attack via an analogous
route is depicted in Scheme 1, Pathway (a) as a possible
mechanism of termination with ammonia. However, the
observed PEtOx ammonia termination deviates from this
expected mechanism and, based on the observed end groups
and byproducts, the mechanism in Scheme 1, Pathway (b) is
proposed. The observed “unconventional” ring opening
derives from nucleophilic attack at the C2 position, causing
CAN bond cleavage which effectively removes the amide
side chain from the nitrogen on the last repeat unit.
Subsequently, the system was end-capped with excess
ammonia and allowed to stir at ambient temperature for
24 h. Interpretation of obtained 1H NMR spectra [Fig. 5(b)]
indicates generation of the proposed hydroxyethylamino
“end group,” with the initiator methyl peak at 2.41 ppm [Fig.
5(b)—a: 2.89H, s], and ethyl –CH2– signals at 2.73 ppm [Fig.
5(b)—b: 1.91H, t] and 3.66 ppm [Fig. 5(b)—c: 1.97H, t].
8
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ACKNOWLEDGMENTS
Subsequent removal of propionitrile from the chain end
yields the alcohol of the hydroxyethylamino end group. Pre-
cedence for nucleophilic attack at the oxazolinium C2-
position had been reported previously, both in the case of
oxazoline CROP end-capping74 and monomeric oxazolinium
salt quenching;67,75,76 however, these reports had been limit-
ed to the addition of water or hydroxide.
PD and GP thank Jessie Bach for performing the NMR measure-
ments, and Ty Londo for running the GPC and GC-MS measure-
ments. KAK and SMG acknowledge the National Science
Foundation for support (CHE-1412439), MALDI-TOF MS
instrumentation (CHE-0619770 (MRI)), and a graduate fellow-
ship (KAK, Bioinnovation IGERT, DGE-1144646).
Given the alternative routes reported for attaching an amino
end group since the first publication of this ammonia termi-
nation protocol, it is presumed that this ammonia termina-
tion method reported by Kobayashi has not been
successfully reproduced by others to yield high purity
amino-terminated POx. The available analytical techniques
for end group characterization at the time of this first publi-
cation—i.e., NMR, IR and titration analysis—would provide
similar results for the hydroxyethlamino end group as would
be expected for the primary amine end group. Additionally,
subsequent post-polymerization modification chemistry
applied to either the amino or the hydroxyethylamino end-
groups are expected to yield similar results. For example, a
recent report by Morgese, et al., investigating the effect of
POx topology on interfacial properties.77 Linear PEtOx was
quenched with ammonia/THF solution and subject to a 2-
step nitrocatechol group functionalization, providing a suc-
cessful synthetic strategy to yield surface-anchored PEtOx,
although the exact chemical nature of the linking group
remains ambiguous.
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