Oxime functionalization strategy for iodinated poly(epsilon-caprolactone) X-ray opaque materials

Article abstract of DOI:10.1002/pola.27706

Since two of the most common technologies for imaging the human body are X-ray radiography and computed tomography (CT), researchers are focused on developing biodegradable and biocompatible polymeric molecules as an alternative to the traditional small m

Full text of DOI:10.1002/pola.27706

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Oxime Functionalization Strategy for Iodinated Poly(epsilon-caprolactone)
X-ray Opaque Materials
Samantha E. Nicolau,1 Lundy L. Davis,1 Caroline C. Duncan,1 Timothy R. Olsen,2
Frank Alexis,2,3 Daniel C. Whitehead,4 Brooke A. Van Horn1
1Department of Chemistry and Biochemistry, College of Charleston, 66 George St., Charleston, South Carolina 29424
2Department of Bioengineering, Clemson University, 203 Rhodes Research Center Annex, Clemson, South Carolina 29634
3Institute of Biological Interfaces of Engineering, Department of Bioengineering, Clemson University, Clemson, South Carolina
29634-0905
4Department of Chemistry, Clemson University, 467 Hunter Laboratories, Clemson, South Carolina 29634
Correspondence to: B. A. Van Horn (E-mail: vanhornba@cofc.edu)
Received 16 March 2015; accepted 13 May 2015; published online 16 June 2015
DOI: 10.1002/pola.27706
ABSTRACT: Since two of the most common technologies for
imaging the human body are X-ray radiography and computed
tomography (CT), researchers are focused on developing bio-
degradable and biocompatible polymeric molecules as an
alternative to the traditional small molecule contrast agents.
This report highlights the synthesis of novel biodegradable
iodinated poly(e-caprolactone) copolymers by oxime “Click”
ligation reactions. A series of ketone-bearing materials are built
by tin (II)-mediated ring-opening polymerization followed by a
postpolymerization deprotection step. The intended X-ray
opacity is imparted through acid-catalyzed oxime postpolyme-
rization modification of the resultant polymers with an iodin-
ated hydroxylamine. All small molecules and polymeric
materials are characterized using proton nuclear magnetic res-
onance (NMR) for purity, functional group stoichiometry, and
number-averaged molecular weight calculations. Additionally,
the polymers are evaluated with gel permeation chromatogra-
phy (GPC) to determine polymer sample polydispersity and
general molecular weight distribution shapes and by differen-
tial scanning calorimetry (DSC) and thermogravimetric analysis
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(TGA) for thermal properties.
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KEYWORDS: biodegradable; iodine; oxime; polyesters; X-ray
materials, whether for circulation in the body or in implant-
able devices.
INTRODUCTION Biomedical imaging technologies can be
used for both diagnostic and therapeutic purposes, thus
making imaging science a critical part of the success of a
patient treatment plan in the applied clinical setting. The
technologies that find broad use are generally either non- or
minimally invasive,1–19 and among those, X-ray and com-
puted tomography (CT) imaging are the most common and
broadly available. Many of the currently utilized X-ray and
CT imaging agents are injectable small molecules with cova-
lently bound iodine that allow for high X-ray attenuation but
only when the contrast molecule is in locally high concentra-
tion. Nonetheless, these small molecules suffer from non-
specific and not easily defined residence in the blood pool
and tissues, and experience rapid clearance from circulation.
Also, they often have to be administered in high doses to
produce significant imaging capability. Our research aims to
overcome these limitations by covalently bonding iodine-
bearing species onto copolymers through postpolymerization
reactions as a new design for biodegradable X-ray contrast
There are many parallel strategies being undertaken to
address the challenge of preparing well-defined X-ray opaque
materials that have controllable and/or predictable biodistri-
bution. Some current investigations include the “packaging”
of the contrast agent as separate small molecules within sta-
bilized organic structures including conventional liposomes,
micelles, and emulsions.20–25 Unfortunately, these methods
of imparting contrast to the material can still suffer from the
"leakage" of the contrast agent from the material over time.
Other strategies have focused on the covalent attachment of
iodine or iodine-containing molecules to the polymer chains,
particles, or matrix.26–32 Various polymeric structures and
architectures such as dendrimers, linear, block, graft, and
hyperbranched polymers are also being investigated. These
materials can differ in the placement of the radiolabels, with
some having the contrast within main chain/backbone of the
polymer,33–37 at the chain end,38 or as a side group on the
Additional Supporting Information may be found in the online version of this article.
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monomer39,40 or grafted unit. However, there are limited reports
of fully biocompatible and biodegradable materials with suffi-
cient X-ray opacity to meet the clinical need of the imaging
community.32,35,41–44 Our approach seeks to achieve a functional
X-ray opaque material that is biodegradable and biocompatible
from a combination of copolymerization and postpolymerization
reactions. For this reason, we turned our attention to aliphatic
polyesters as our polymeric backbone of choice.
Aliphatic polyesters like poly(e-caprolactone), poly(glycolic
acid), and poly(lactic acid) have demonstrated biocompatibil-
ity and biodegradability and continue to be a standard poly-
mer backbone for drug delivery systems and other
biomedical applications.45,46 Nevertheless, one major limita-
tion to polyester systems is the fact that the ring-opening
polymerizations of the commercially available lactones and
lactides normally result in polymers that are not functional
and cannot be easily modified. This drawback can be com-
batted with the incorporation of a second monomer via a
copolymerization strategy.47–56 By using the simple lactone
along with another functional lactone, the polyester system
becomes a scaffold onto which many different types of small
molecules can be attached through postpolymerization, as
depicted in Figure 1. It is the goal of this research to present
a new type of X-ray opaque material that is (1) quickly syn-
thesized, (2) modular, and (3) inherently biodegradable.
FIGURE 1 General postpolymerization strategy for functional-
ized copolymers.
yphthalimide, triethylamine, and hydrazine monohydrate
were purchased from Sigma Aldrich and used as received.
Anhydrous sodium sulfate, sodium bisulfite, and sodium
bicarbonate were purchased from Fisher Scientific and used
as received. Toluene (Sigma Aldrich) was dried by heating at
reflux over sodium and distilled under nitrogen prior to use.
All other solvents (ethyl acetate, hexanes, methanol (MeOH),
dichloromethane (CH2Cl2), deuterated chloroform (CDCl3),
and tetrahydrofuran (THF)) were used as received. e-
Caprolactone (CL, Sigma Aldrich) and benzyl alcohol (BnOH,
Sigma Aldrich) were distilled from calcium hydride (CaH2)
and stored under nitrogen prior to use. Tin (II) 2-
ethylhexanote (Sn(Oct)2, Sigma Aldrich) was stored over 4–6
Å molecular sieves prior to use. Para-toluenesulfonic acid
monohydrate (TsOH, Sigma Aldrich) was dissolved in THF to
afford a 0.02 M solution.
Many methodologies are being employed for postpolymeriza-
tion modification, with “Click” chemistries of all types receiv-
ing significant attention. These reactions meet the criteria of
being (1) modular (as in able to work on a variety of sub-
strates and without much ligand specificity), (2) high-yielding,
(3) produce harmless side products, and (4) are attainable
with mild reaction conditions. Oxime chemistry has broad
application as a “Click” reaction in biomolecule conjugation
due to its favorable reaction kinetics and thermodynamics
under near physiological conditions. It is also a promising
polymer conjugation reaction with many examples appearing
in the recent synthetic polymer literature, some of which uti-
lize polyester backbones.57–75 This versatility is exploited in
our work in the preparation of ketone-functionalized polyest-
ers capable of reacting with hydroxylamines to provide a
covalent linkage between the polymer and contrast agent. Our
interest in the oxime conjugation partly stems from the syn-
thetic simplicity in preparing aminooxy derivatives, including
the desired O-(2-iodobenzyl) hydroxylamine used to impart
the desired X-ray opacity, while maintaining the biodegrad-
ability and potentially the biocompatibility of the polyester
material itself. Herein, we describe our syntheses and subse-
quent conjugation of the iodinated species to create a func-
tional copolymer. Furthermore, we provide characterization of
the structure, size, and thermal properties of a novel proposed
X-ray opaque material with inherent biodegradability.
Instrumentation and Measurements
Proton and carbon nuclear magnetic resonance (1H and 13C
NMR) spectroscopy experiments were conducted using a
300 MHz Varian Mercury 300 Vx NMR spectrometer. Samples
were acquired in deuterated chloroform for nt532 or 128
for proton and nt51024 or 4096 for carbon experiments of
small molecules and polymers, respectively. Basic processing
and storage were achieved on a Sun Microsystem worksta-
tion. NMR figures were generated using Spinworks freeware
to process the FID and then exported for plotting using
Origin 7.0.
GPC data were acquired on a Malvern GPCMax equipped
with an external column heater (35 8C) and Viscotek refrac-
tive index detector (VE3580) using inhibited THF as an elu-
ent. Samples were prepared at 1.0 mg/mL in THF and
filtered through 0.2 lm PTFE syringe filters (VWR Interna-
tional). Separation was achieved through use of the following
columns in series: Malvern (CLM3008-Tguard) Organic Guard
Column (10 mm x 4.6 mm), Waters Styragel HR 4ETHF, and
Malvern (T6000M) General Mixed Bed (300 mm x 7.8mm)
over a 40 minute sample run with molecular weights and
polydispersity calculated from a third-order calibration curve
from twelve different polystyrene standards Mp ranging from
1050–3.8x106 Da.
EXPERIMENTAL
Materials
Meta-chloroperoxybenzoic acid (m-CPBA), 1,4-cyclohexan-
dione monoethylene ketal, 2-iodobenzylbromide, N-hydrox-
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IR spectra were recorded on Bruker Alpha FT-IR spectrome-
ters using Opus 6.5 software.
followed by 45 mL of THF. Triethylamine (2.8 mL, 20.1
mmol, 1.5 eq.) was added to the reaction flask using a 5 mL
syringe and a red color was immediately observed upon
addition. A stock solution of 2-iodobenzyl bromide (3.97 g,
13.4 mmol, 1.0 eq.) in THF (15 mL) was added dropwise to
the reaction round bottom flask in 3 aliquots of 5 mL. The
flask was capped and the reaction was allowed to proceed at
room temperature for 18 h. The crude reaction mixture was
characterized with TLC using a 1:1 hexanes:ethyl acetate elu-
ent. After removal of the THF solvent by rotary evaporation,
the reaction mixture contents were transferred into a separa-
tory funnel by rinsing of the round bottom flask with meth-
ylene chloride (165 mL) and water (165 mL). After initial
separation, the organic layer was set aside and the aqueous
layer was extracted 2 x 100 mL of CH2Cl2. The organic layers
were combined and then washed with distilled water (3 x
100 mL) and once with brine (100 mL). The combined
organic layer was then dried over anhydrous sodium sulfate
in a 500 mL Erlenmeyer overnight. The organic layer was fil-
tered the following day and concentrated by rotary evapora-
tion and high vacuum to yield an off-white powdery solid.
No further purification by column chromatography was
required. Yield: 4.72 g (93 % isolated). 1H NMR (300 MHz,
CDCl3, d): 7.92 (m, 1H, Ar H), 7.90 (m, 1H, phthalimido),
7.78 (m, 1H, phthalimido), 7.61 (d, 1H, Ar H), 7.48 (t, 1H, Ar
H), 7.01 (t, 1H, Ar H), 5.35 (s, 2H, C2H4ICH2-) ppm. 13C NMR
(75 MHz, CDCl3, d): 163.6 (phthalimide), 139.8, 137.1, 134.7,
131.4, 130.1, 129.1, 129.7, 123.8, 99.8, 83.1 ppm; IR (solid,
ATR): m 5 3057 (w), 2962-2854 (w), 1783 and 1723 (vs,
broad over range to 1650), 1618 (w), 1607 (w), 1586 (w)
1462 (m), 1439 (m), with fingerprint peaks at 1387, 1370,
Differential Scanning Calorimetry (DSC) experiments were
conducted on a Perkin Elmer DSC 7 over a range of 220 to
180 8C at 5 8C per minute. The data were then processed
using the Pyris software to obtain Tm values. Thermogravi-
metric Analysis (TGA) was performed on a TA Instruments
Hi-Res TGA 2950 thermogravimetric analyzer by running
samples from 20 to 600 8C at 10 8C per minute under
nitrogen.
X-ray imaging was performed using a Tingle 325MVET x-ray
machine with 51 kVp, 300 mA, and 5 millisecond exposure
time. X-ray samples were prepared from ꢀ 10 mg each of a
control sample of polylactide (PLA), the synthesized P(CL-co-
OPD) copolymer, 5a, and its corresponding functionalized
copolymer, 6a, where the iodine content of the polymer is
calculated to be 11.4 weight % iodine after conjugation. PLA
was selected as a control because it is commonly used for
biomedical applications and does not have radio-opaque
properties.
Synthesis
1,4,8-Trioxaspiro[4.6]-9-undecanone (1, TOSUO)
[This procedure is a modification of the synthesis originally
reported.76–79
] 1,4-cyclohexanedione monoethylene acetal
(4.99 g, 32.0 mmol, 1.0 eq.) was dissolved in methylene chlo-
ride (50 mL) in a 300 mL RBF and was allowed to stir for
10 minutes. Meta-chloroperoxybenzoic acid (11.50 g, 48.0
mmol, 1.5 eq.) was added to the flask in scoops to the
300 mL round bottom flask over 30 minutes. A white precip-
itate was noted approximately 20 minutes after all reagents
had been added. The reaction was allowed to proceed with
stirring at room temperature overnight. The contents of the
reaction flask were added to a 1000 mL Erlenmeyer flask
equipped with a stirbar, followed by 100 mL of H2O and
50 mL of CH2Cl2. Sodium bisulfite (7.67 g) was then added
to the stirring mixture over 30 minutes, followed by sodium
bicarbonate (6.82 g), and allowed to stir overnight. The con-
tents of the Erlenmeyer were then poured into a 2 L separa-
tory funnel where the organics were collected. The aqueous
layer was washed with 2 x 50 mL of CH2Cl2. The combined
organic layers were then extracted with 2 x 50 mL of sodium
bisulfite solution, 2 x 50 mL of saturated sodium bicarbonate
solution, and 1 x 100 mL of brine. The organic layer was
then dried over anhydrous sodium sulfate and concentrated
by rotary evaporation to yield a viscous off-white oil that
became a crystalline white solid under high vacuum. Yield:
4.91 g (89 %) 1H NMR (300 MHz, CDCl3, d): 4.25 (m, 2 H,
-COOCH2-), 3.94 (s, 4H, acetal -OCH2CH2O-), 2.67 (m, 2H,
-COCH2-), 1.98 (m, 2H, -COOCH2CH2-), 1.87 (m, 2H,
-COCH2CH2-); 13C NMR (75 MHz, CDCl3, d): 175.7 (C5O),
108.1 (ketal), 65.0, 64.6, 39.3, 32.9, 29.1 ppm.
1354, 1183, 1128, 1102, 1079, 1011, 967, 875 cm21
.
O-(2-iodobenzyl) Hydroxylamine (3)
O-(2-iodobenzyl)-N-hydroxyphthalimide (0.50 g, 1.32 mmol,
1.0 eq.) was massed into a 100 mL round bottom flask
equipped with a stirbar. To this flask, THF (15 ml) was
added and the mixture was allowed to stir for 15 minutes to
dissolve the starting material. Hydrazine monohydrate
(0.35 mL, 7.2 mmol, 5.5 eq.) was then added by syringe to
the reaction and a light yellow color change was observed.
The reaction was allowed to proceed for 24 h at room tem-
perature. Next, the reaction mixture (murky white) was
washed twice with water, once with brine, and once with
methylene chloride. The mixture was purified by column
chromatography with methylene chloride as eluent (increas-
ing polarity with methanol as needed) and concentrated by
rotary evaporation to afford an off-white oil. Yield: 0.32 g
1
(97% isolated). H NMR (300 MHz, CDCl3, d): 7.81 (d, 1H, Ar
H), 7.35-7.49 (m, 2H, Ar H), 7.0 (t, 1H, Ar H), 6.51 (broad s,
2H, -ONH2), 4.69 (s, 2H, C6H4ICH2-) ppm; 13C NMR (75 MHz,
CDCl3, d) 139.9, 139.7, 129.8, 128.5, 99.1, 81.7 ppm; IR
(from CDCl3 solution): m 5 3309 and 3235 (m, broad), 3146
(w), 3059 (w) 2920 and 2867 (w, broad), 1584, 1563, 1464,
and 1436 (m), with fingerprint peaks at 1272, 1184, 1109,
O-(2-iodobenzyl)-N-hydroxyphthalimide by Nucleophilic
Substitution from 2-iodobenzyl Bromide (2)
N-hydroxyphthalimide (2.86 g, 17.53 mmol, 1.3 eq.) was
added to a 500 mL round bottom flask using a solids funnel
1045, 1006, 944, 900, 745, 648 cm21
.
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Example Procedure for Conventional Ring-opening
Polymerization Using CL, TOSUO, Sn(Oct)2, and Benzyl
Alcohol to Afford Poly(CL71-co-TOSUO11) (4a)
Example Procedure for the Oxime “Click” Reaction of O-
(2-iodobenzyl)hydroxylamine onto P(CL71-co-OPD11) to
Afford Functionalized Copolymer P(CL71-co-(OPD-mod-(2-
IBn))11) (6a)
Dry CL (6.6 mL, 60 mmol, 90 eq.) and TOSUO (3) (1.19 g,
6.9 mmol, 10 eq.; from a 2.0 M dry toluene solution) were
added to a 100 mL 3-neck round bottom flask equipped
with a stirbar using dry syringes and needles. An additional
4 mL of dry toluene was added to the reaction flask under
inert N2 atmosphere, followed by distilled benzyl alcohol (70
lL, 0.66 mmol, 1.0 eq.) and tin (II) octanoate catalyst (110
lL, 0.34 mmol, 0.51 eq.). The bottom of the flask was sub-
merged in a silicone oil bath with the temperature set to
maintain 110 8C. Reaction was monitored by removal of an
aliquot for 1H NMR analysis at 18 h and was subsequently
quenched with 2 drops of TsOH (0.2 M in THF). The reaction
mixture was precipitated in 1500 mL of cold methanol to
yield white solid that was collected on a fritted funnel and
dried under vacuum. Yield: 6.72 g (87% overall yield as meas-
ured from 96% conversion of e-CL and 94% conversion of
P(CL71-co-OPD11) polymer (0.203 g, 0.021 mmol polymer
containing 0.23 mmol ketone) was massed into a scintillation
vial equipped with a stirbar and to it was added 3 mL of
THF. A 10 mL stock solution of O-(2-iodobenzyl) hydroxyla-
mine (0.10 M) was prepared in a different vial and 2.35 mL
of hydroxylamine stock were subsequently delivered by
syringe to the reaction vial. Three drops of a THF stock solu-
tion of TsOH (0.02 M) were added to the reaction vial and
the reaction was allowed to proceed with stirring for 24 h at
room temperature. The contents of the vial were then pre-
cipitated into 300 mL cold hexanes, followed by collection
by filtration and drying under vacuum. Yield: 0.177 g (79%
isolated) Abbreviated as P(CL71-co-(OPD-mod-(2-IBn))11)
1H
NMR (300 MHz, CDCl3, d): 7.83-7.81 (dd, 1H, Ar H3), 7.4–
7.35 (m, 5H, Ar H end group), 7.35-7.31 (dd and td, 2H, Ar
H3 & H5), 6.98 (td, 1H, Ar H4), 5.12 (s, 2H, benzylic H), 5.1
(d, 2H, -CH2ON- oxime), 4.27 (m, 2H, -CH2OCO- oxime), 4.05
(t, 2H, -CH2OCO- CL), 3.65 (t, 2H, -CH2OH end group), 2.70-
2.45 (three t, 6H, OCOCH2CH2C(oxime)CH2CH2O- OPD), 2.30
(t, 2H, -OCOCH2- CL), 1.60 (m, 4H, -OCOCH2CH2CH2CH2-
CH2O- CL), 1.40 (m, 2H, -OCOCH2CH2CH2CH2CH2O- CL) ppm.
13C NMR (75 MHz, CDCl3, d) 173.8, 173.5, 172.9, 157.1,
156.3, 140.5, 139.5, 139.4, 129.52, 129.48, 128.3, 98.2, 98.1,
79.5, 64.8, 64.4, 34.3, 34.2, 34.0, 30.5, 28.6, 25.8, 24.8 ppm;
1
TOSUO by H NMR of the crude reaction mixture). Confirmed
final product as poly(CL71-co-TOSUO11). 1H NMR (300 MHz,
CDCl3, d): 7.35-7.4 (m, 5H, Ar H), 5.12 (s, 2H, benzylic H of
end group), 4.15 (m, 2H, -CH2OCO- TOSUO), 4.05 (t, 2H, -
CH2OCO- CL), 3.95 (s, 4H, -OCH2CH2O- TOSUO ketal), 3.65 (t,
2H, -CH2OH end group), 2.39 (t, 2H, -OCOCH2- TOSUO), 2.30
(t, 2H, -OCOCH2- CL), 2.05-1.90 (m, 4H, - OCOCH2CH2C(OCH2-
CH2O)CH2CH2O- TOSUO), 1.60 (m, 4H, -OCOCH2CH2CH2CH2-
CH2O- CL), 1.40 (m, 2H, - OCOCH2CH2CH2CH2CH2O- CL) ppm.
13C NMR (75 MHz, CDCl3, d) 173.8, 173.6, 128.8, 128.4, 109.6,
77.5 (not CDCl3) 65.3, 64.5, 64.3, 62.7, 60.5, 60.4, 36.2, 34.4,
34.3, 32.8, 32.5, 28.9, 28.8, 28.5, 25.7, 25.5, 24.9, 24.8, 24.7
ppm; Tm, DSC 5 44.7 8C (range 40.1–46.3 8C)
Tm, DSC 5 37.4 and 42.7 8C (range 26.9–44.1 8C)
RESULTS AND DISCUSSION
Small Molecule Syntheses of Monomer and Iodinated
Hydroxylamine
Example Procedure for the Polymeric Ketal Deprotection
Using Trityltetrafluoroborate to Afford of Poly(CL71-co-
OPD11) (5a)
To allow for the additional chemical functionality to be
imparted to the polyester material, we chose to use a copoly-
merization and postpolymerization strategy. Our devised
route employs a previously published monomer, 1,4,8-triox-
aspiro[4.6]-9-undecanone (abbreviated TOSUO, 1) which is
prepared from the Baeyer-Villiger oxidation of 1,4-cyclohexa-
nedione monoethylene acetal. This functional monomer has
been reported previously to copolymerize with CL77–80 and
was utilized in our desired polymer syntheses.
P(CL71-co-TOSUO11) (1.98 g, 0.196 mmol, 1.0 eq. of polymer
with 11.0 eq. of ketone) was transferred into a 500 mL
round bottom flask followed by 200 mL of CH2Cl2. Tritylte-
trafluoroborate (0.94 g, 2.8 mmol, 1.3 eq. per ketone) was
added to the stirring flask and a bright yellow/orange color
was observed. The reaction was allowed to proceed for 1 h.
The reaction mixture was added by pipette into 1500 mL of
ice cold methanol and allowed to stir for > 3 h. The white
solid product was isolated over a fritted funnel and dried
with vacuum. Yield: 1.40 g. (74% isolated) 1H NMR (300
MHz, CDCl3, d): 7.35-7.4 (m, 5H, Ar H end group), 5.12
(s, 2H, benzylic H), 4.35 (m, 2H, -CH2OCO- OPD), 4.05 (t, 2H,
-CH2OCO- CL), 3.65 (t, 2H, -CH2OH end group), 2.80-2.75
(two t, 4H, OCOCH2CH2COCH2CH2O- OPD), 2.39 (t, 2H,
-OCOCH2- OPD), 2.30 (t, 2H, -OCOCH2- CL), 1.60 (m, 4H,
-OCOCH2CH2CH2CH2CH2O- CL), 1.40 (m, 2H, -OCOCH2CH2
CH2CH2CH2O- CL) ppm. 13C NMR (75 MHz, CDCl3, d) 206.0,
173.7, 173.5, 172.9, 128.8, 128.4, 77.5 (not CDCl3), 64.7,
64.3, 62.7, 59.4, 59.3, 41.7, 37.6, 34.3, 34.1, 33.6, 32.5, 28.54,
28.47, 28.0, 25.72, 25.67, 25.5, 24.8 24.6 ppm; Tm, DSC 5 57.1
8C (range 55.4–58.4 8C)
The preparation of the iodine-containing hydroxylamine was
achieved through the nucleophilic substitution of 2-
iodobenzyl bromide by N-hydroxyphthalimide in the pres-
ence of triethylamine also shown in Scheme 1. The removal
of phthalimido group was accomplished by exposing the
phthalimido derivative, 2, to hydrazine overnight at room
temperature to yield a final product of O-(2-iodobenzyl)
hydroxylamine, 3, after a short column purification. Both the
purified O-(2-iodobenzyl)-N-hydroxyphthalimide, 2, and final
1
O-(2-iodobenzyl) hydroxylamine, 3, were characterized by H
and 13C NMR spectroscopies, which confirmed the disappear-
ance of the second aromatic resonances and shifting of the
benzylic methylene unit adjacent to the hydroxylamine as
visualized in the proton NMR spectrum and aromatic inset
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SCHEME 1 Small molecule syntheses.
of Figure 2. Infrared spectroscopy also confirmed the struc-
tures with carbonyl absorbances at 1783 and 1723 cm21
indicative of the phthalimido group that were then absent in
the hydroxylamine, where additional N-H stretches at 3309
and 3235 cm21 were observed.
Polymer Synthesis and Ketal Deprotection
Traditional ring-opening polymerization with tin (II) octano-
ate was employed to create copolymers with a predictable
incorporation of comonomer, as represented in the Scheme 2.
SCHEME 2 Ring-opening polymerization, deprotection, and
functionalization reactions.
This copolymerization of CL and TOSUO was achieved using
benzyl alcohol as the initiator in dry toluene at 20 weight per-
cent monomer at 110 8C for 18 h. The reaction was monitored
1
with H NMR spectroscopy by calculating the percent conver-
sion of monomer to polymer with the methylene unit ratios
on the oxygen side of the ester with measured con-
versions > 90% for all polymerizations. The final products
were isolated from precipitation in methanol non-solvent,
dried under vacuum and then characterized with 1H NMR
spectroscopy to determine a percent TOSUO incorporation
and a number average molecular weight as tabulated in Table
1. Subsequent removal of the ketal units was achieved using
trityltetrafluoroborate in dichloromethane, followed by pre-
cipitation in methanol, and isolation and drying of the solid
product to yield poly(caprolactone-co21,4-oxepan-1,5-dione),
abbreviated P(CL-co-OPD). Figure 3 provides representative
1H NMR spectra of purified samples 4b and 5b with the incor-
poration of TOSUO monomer calculated at 17.0 mol% as
FIGURE 2 Overlay of 1H NMR spectra comparing the hydroxyl-
amine synthetic products.
P(CL78-co-TOSUO16) and its corresponding P(CL78-co-OPD16)
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TABLE 1 Proton NMR and GPC Characterization of Copolymers and Modified Copolymers
CL Repeat
Unitsa
Functional
Repeat Unitsa
Mole %
PDIb
a
b
b
Polymer Sample
Functionality
Mn, NMR
Mn, GPC
Mw, GPC
4a P(CL-co-TOSUO)
71
71
71
78
78
78
11
11
11
16
16
16
13.1
13.1
13.1
17.0
17.0
17.0
10100
9620
10800
13200
13600
19000
18900
19900
17300
17900
18400
29200
27800
28400
1.60
1.36
1.35
1.54
1.47
1.43
5a P(CL-co-OPD)
6a Iodine Modified Copolymer
4b P(CL-co-TOSUO)
12200
11800
11100
14800
5b P(CL-co-OPD)
6b Iodine Modified Copolymer
a
b
Calculated from 1H NMR spectra using the ratio of the –COOCH2-
methylene integrations of CL and TOSUO repeat units in the polymers
and the benzylic methylene end group after isolation from MeOH
precipitation.
GPC data acquired with a Malvern GPCMax with an RI detector and
PS standards from single runs on same day.
deprotected ketone polymer daughter product. These spectra
confirm the complete removal of the ethylene glycol units and
shifting of the methylene units alpha to the ketones. Addition-
ally, 13C NMR spectroscopy established the presence of ketone
units at 206 ppm in the OPD polymer and the disappearance
of the spiroketal carbon at 109.6 ppm after the deprotection
step.
lents of hydroxylamine per ketone under the conditions
listed above.
Proton NMR spectroscopy confirmed that ꢀ100% coupling
was achieved on both samples (Products 6a and 6b) as can
be seen by the complete shifting of the two different methyl-
ene subunits alpha to the ketone (from b’, d’ to b’’, d’’) to
new positions in the oxime product in Figure 4 along with a
shift in the methylenes (from e’ to e’’) adjacent to the oxygen
of the backbone ester groups. Additionally, new benzylic
methylene resonances (f’’) appear at 5.1 ppm. These NMR
results provide evidence of a well-defined and controllable
coupling reaction stoichiometry as observed through charac-
terization of the final products. 13C NMR also indicated
attachment of the hydroxylamine through the appearance of
new aromatic resonances from 99–140 ppm, appearance of a
pair of oxime isomer resonances at 156.3 and 157.1 ppm, as
well as the disappearance of the C5O resonance at 206
ppm. The GPC overlay in Figure 5 and data tabulated in
Postpolymerization Modification via Acid-catalyzed
Oxime Chemistry
Attachment of the newly synthesized O-(2-iodobenzyl)
hydroxylamine to the P(CL-co-OPD) polymer backbones was
achieved through p-toluene sulfonic acid-catalyzed oxime for-
mation in THF solution, followed by precipitation into cold
methanol, isolation by filtration, and drying under vacuum to
yield a white solid graft copolymer. To demonstrate the
reproducibility and effective matching of reaction mixture
and polymer product stoichiometries, each of the ketone-
bearing polymers (5a and 5b) was exposed to 1.1 equiva-
FIGURE 3 1H NMR spectral overlay of polymer deprotection of
FIGURE 4 1H NMR comparison of P(CL-co-OPD) 5b and P(CL-
P(CL-co-TOSUO) 4b to yield P(CL-co-OPD) 5b.
co-(OPD-mod-(2-IBn))) 6b.
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modified PCL material, thermal analysis by differential scan-
ning calorimetry (DSC) was performed on each of the poly-
mer precursors and the final products. As expected, both the
P(CL-co-TOSUO) initial copolymer 4a and the oxime graft
product 6a display lower melting transition temperatures
[Fig. 6(a)] than unfunctional pure PCL (Tm ꢀ 60 8C) while
the P(CL-co-OPD) 5a melts at higher temperatures. These
results are expected due to the disruption of the crystalline
packing of the polymers arising from the spiroketal and
bulky aromatic side chains on the P(CL-co-TOSUO) and oxime
graft product, respectively, and the increased regular packing
and improved crystalline structure with the intermediate
ketone-bearing OPD polymer.81–83 The lower Tm range (35
8C > Tm > 50 8C) for the final graft copolymers is particularly
interesting since a material Tm near physiological tempera-
tures could have a significant impact on the material degra-
dation in vivo.
To learn more about how the compositional and structural
changes of the oxime conjugated copolymer affect the ther-
mal stability of the system, thermogravimetric analysis (TGA)
was performed. Main chain PCL degradation and depolymer-
ization were observed for the OPD and oxime copolymers at
temperature ꢁ 400 8C as expected while the starting ketal
copolymers degraded as a whole at significantly lower tem-
peratures, as depicted in Figure 6(b). Interestingly, we did
not observe much of an early thermal degradation mode for
the P(CL-co-OPD) polymers such as 5a in the 150–250 8C
region. This degradation mode is believed to represent the
b-elimination mechanism resulting from the methylenes
adjacent to the ketone or oxime units. Degradation of this
type has been previously documented for ketone-containing
polymer systems81 as well as oxime-modified polymers, and
begins at temperatures as low as 150 8C. For the oxime-
modified copolymers (see Supporting Information Figure
S1 - derivative plot and Figure 6(b)), including polymer 6a,
a substantial mass loss was detected and peaked at about
FIGURE 5 Overlay of GPC curves of copolymers.
Table 1 of the 4a, 5a, and 6a products confirm that no
unwanted degradation of the polymer backbone was
observed during the 24 h exposure to the acid catalyst. Addi-
tionally, no dramatic change in the molecular weight distri-
bution or peak molecular weight of the chromatogram for
the modified copolymer was observed relative to the ketone-
bearing and ketal-containing polymer precursors. These
NMR and GPC results support our claim of the stability of
the polymer under varying reaction conditions and the effi-
ciency and accuracy of the polymer oxime reaction for creat-
ing iodinated poly(e-caprolactone) materials.
Thermal Analysis of Copolymers and Graft Copolymers
with DSC and TGA
Given the influence of thermal stability and crystallinity on
the potential in vivo degradation of the synthetic iodine-
FIGURE 6 Thermal analysis of copolymers with (a) DSC and (b) TGA.
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ACKNOWLEDGMENTS
This work has been supported by multiple College of Charles-
ton sources to provide undergraduate researcher and faculty
stipends, supplies, and instrument time. The internal sources
include start-up funds, the Department of Chemistry and
Biochemistry, and the Undergraduate Research and Creative
Activities office (SURF awards including SU2013-011 and
SU2013-035; RPGs including RP2013-007, RP2013-010,
RP2014-010, and RP2014-017). Additionally, this research was
supported in part by Howard Hughes Medical Institute (HHMI)
Undergraduate Science Education Grants to the College of Char-
leston (# 52006290 and #52007537) through four Summer
Research Awards. This material is also supported by the
National Science Foundation under MRI grant CHE-1229559.
We would like to thank Kimberly Ivery (Clemson) for the TGA
analysis and the Institute for Biological Interfaces of Engineer-
ing (IBIOE) for DSC assistance.
FIGURE 7 Preliminary X-ray analysis of (a) a PLA polymer con-
trol, (b) the P(CL-co-OPD) control, 5a, and (c) the oxime-
modified sample, 6a using ꢀ 10 mg samples. X-ray imaging
was performed using a Tingle 325MVET x-ray machine with 51
kVp, 300 mA and 5 millisecond exposure time.
325 8C, potentially due to the removal of the oxime-linked
benzyl units, which correlates well with ꢀ30% reduction in
mass being expected if all OPD-mod-OBnI units were cleaved
in heating for this sample. These data further confirm the
successful covalent attachment of the iodinated benzyl
hydroxylamines to the P(CL-co-OPD) polymers.
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