Poly(ethylene glycol)-based ammonium ionenes containing nucleobases

Article abstract of DOI:10.1016/j.polymer.2013.01.040

Adenine-functionalized ionenes (ionene-A) and thymine-functionalized ionenes (ionene-T) were synthesized from novel nucleobase-containing monomers from an efficient aza-Michael addition to an acrylate precursor. Complexes of ionenes with nucleobase-containing guest molecules (n-butyl thymine, nBT and n-butyl adenine, nBA) exhibited well-defined complementary hydrogen bonding interactions. Job's analysis revealed a 1:1 stoichiometry for the hydrogen-bonded complexes of [ionene-A]:[nBT], [ionene-T]:[nBA], and [nBA]:[nBT]. A mathematical fit of the chemical shifts to the Benesi-Hildebrand method revealed the association constants of 94 M^-1, 130 M ^-1, and 137 M^-1 for complexes of [ionene-A]:[nBT], [ionene-T]:[nBA], and [nBA]:[nBT], respectively. Furthermore, DSC thermograms of 1:1 [ionene-A/T]:[guest molecule] complexes showed the disappearance of the melting transition for the guest molecule, confirming the absence of macrophase separation of the nucleobase guest in the polymer matrix. Morphological studies including atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) revealed a microphase-separated morphology for the nucleobase-containing ionenes and less defined microphase separation for the 1:1 complexes.

Full text of DOI:10.1016/j.polymer.2013.01.040

Polymer 54 (2013) 1588e1595
Contents lists available at SciVerse ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Poly(ethylene glycol)-based ammonium ionenes containing nucleobases
*
Mana Tamami, Sean T. Hemp, Keren Zhang, Mingqiang Zhang, Robert B. Moore, Timothy E. Long
Macromolecules and Interfaces Institute, Department of Chemistry, Virginia Tech, Blacksburg, VA 24061, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Adenine-functionalized ionenes (ionene-A) and thymine-functionalized ionenes (ionene-T) were syn-
thesized from novel nucleobase-containing monomers from an efficient aza-Michael addition to an
acrylate precursor. Complexes of ionenes with nucleobase-containing guest molecules (n-butyl thymine,
nBT and n-butyl adenine, nBA) exhibited well-defined complementary hydrogen bonding interactions.
Job’s analysis revealed a 1:1 stoichiometry for the hydrogen-bonded complexes of [ionene-A]:[nBT],
[ionene-T]:[nBA], and [nBA]:[nBT]. A mathematical fit of the chemical shifts to the BenesieHildebrand
method revealed the association constants of 94 Mꢀ1, 130 Mꢀ1, and 137 Mꢀ1 for complexes of [ionene-
A]:[nBT], [ionene-T]:[nBA], and [nBA]:[nBT], respectively. Furthermore, DSC thermograms of 1:1 [ionene-
A/T]:[guest molecule] complexes showed the disappearance of the melting transition for the guest
molecule, confirming the absence of macrophase separation of the nucleobase guest in the polymer
matrix. Morphological studies including atomic force microscopy (AFM) and small-angle X-ray scattering
(SAXS) revealed a microphase-separated morphology for the nucleobase-containing ionenes and less
defined microphase separation for the 1:1 complexes.
Received 14 November 2012
Received in revised form
24 January 2013
Accepted 27 January 2013
Available online 1 February 2013
Keywords:
Segmented ionenes
Nucleobases
1H NMR titration
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
charge of ammonium ionenes, they are versatile in many bio-
medical applications such as gene transfection agents [4,5], cos-
Future advances in polymer design will involve the use of
non-covalent interactions, such as hydrogen bonding, electro-
metics [6], antimicrobial agents [7], and flocculants for water
treatment [8].
statics,
p
e
p
interactions, metal coordination, and Van der Waals
Two general families of ionenes are segmented and non-
segmented copolymers with various topologies. Segmented [9]
ionenes have oligomeric, low Tg spacers between their charged
sites and demonstrate enhanced mechanical properties compared
to non-segmented [10] ionenes, which generally contain shorter
alkylene spacers between charges and therefore higher charge
density along the backbone. In this manuscript, we report seg-
mented 1000 g/mol poly(ethylene glycol) (PEG)-based ionenes that
contain pendant adenine or thymine units. The PEG segment en-
hances polymer solubility in organic solvents and increases chain
mobility compared to non-segmented ionenes, leading to desirable
complementary hydrogen bonding interactions.
Previous literature describes a wide variety of synthetic poly-
mers functionalized with complementary adenine and thymine
nucleobases or with nucleobase mimics [11e17]. However, few
reports detail the incorporation of complementary hydrogen
bonding in the presence of ionic interactions. Rotello et al. [18] used
orthogonal self-assembly of polymers and nanoparticles to micro-
pattern surfaces. However, the hydrogen bonding recognition units
and charged species resided on separate polymer backbones. Weck
et al. [19] synthesized ammonium and 2,6-diamonopyridine (DAP)-
functionalized norbornene diblock copolymers containing both
forces to develop supramolecular polymers for diverse applications.
DNA exquisitely utilizes both complementary hydrogen bonding
with nucleobases and ionic interactions and many polymer scien-
tists draw inspiration from DNA to synthesize nucleobase-
containing structures [1]. Environmental stimuli including tem-
perature, solvent polarity, humidity, concentration, and pH readily
control the strength of these interactions, thus leading to respon-
sive polymers [2].
Ionenes are ion-containing polymers that contain quaternized
nitrogens in their backbone [3]. The quantitative step-growth pol-
ymerization of a ditertiary amine with a dihalide monomer pro-
duces families of ionenes. Typical monomers include dimethyl
amines and alkyl chlorides to ensure a quantitative polymerization
process for high molecular weights. The synthetic strategy provides
control over charge density and placement, and consequently
ionenes are ideal models to investigate structure-property re-
lationships of well-defined cationic polymers. Due to the positive
* Corresponding author.
E-mail address: telong@vt.edu (T.E. Long).
0032-3861/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.polymer.2013.01.040

M. Tamami et al. / Polymer 54 (2013) 1588e1595
1589
electrostatic and hydrogen bonding interactions. They concluded
from 1H NMR titrations that the non-covalent interactions were
orthogonal, and electrostatics did not disrupt hydrogen bonding.
Recently, Hemp et al. [20] copolymerized 9-vinylbenzyladenine
(VBA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) with
subsequent protonation to provide adenine-containing poly-
electrolytes. Copolymers having 11, 22, and 35 mol% of VBA showed
polyelectrolyte behavior in dilute and semidilute regimes. Due to
the hydrogen bonding association between adenine units, they did
not observe polyelectrolyte behavior in the concentrated regime. In
addition, the electrospinning behavior showed a strong depend-
ence on the VBA incorporation. We also demonstrated the interplay
between hydrogen bonding and electrostatic interactions in both
solid and solution states with poly(9-vinylbenzyladenine-b-n-butyl
acrylate-b-9-vinylbenzyladenine) triblock copolymers and selec-
tive association of uracil-containing salts with adenine units [21].
We report the synthesis and characterization of water-soluble,
adenine (A) and thymine (T) functionalized (KAT ca. 100 Mꢀ1 in
CDCl3) segmented poly(ethylene glycol) (PEG)-based ammonium
ionenes for supramolecular assembly. Supramolecular assembly
studies between the adenine and thymine nucleobases probed the
influence of electrostatics on complementary hydrogen bonding. In
the solution state, 1H NMR titration studies determined the stoi-
chiometry as well as association constants, Ka, between adenine-
and thymine-containing PEG-ionenes with complementary guest
molecules. In the solid state, we investigated the effect of self-
complementary hydrogen bonds and complementary hydrogen
bonds on thermal and morphological properties of adenine- and
thymine-ionenes as well as their complexes with guest molecules.
equipped with Nanosensor silicon tips having a spring constant of
42 N/m. Samples were imaged at a set-point ratio of 0.70 with
a
magnification of
1
m
m ꢂ 1
mm. SAXS was performed using
a Rigaku S-Max 3000 3 pinhole SAXS system, equipped with
a rotating anode emitting X-ray with a wavelength of 0.154 nm (Cu
Ka). Scattering from a Silver behenate standard was used to cali-
brate the sample-to-detector distance. For SAXS, the sample-to-
detector distance was 1603 mm. Two-dimensional SAXS patterns
were obtained using a fully integrated 2D multiwire, proportional
counting, gas-filled detector, with an exposure time of 1 h. All SAXS
data were analyzed using the SAXSGUI software package to obtain
radically integrated SAXS intensity versus scattering vector q
(SAXS), where q ¼ (4
p/l)sin(q), q is one half of the scattering angle
and is the wavelength of X-ray profiles.
l
2.3. Synthesis of N,N-bis(3-(dimethylamino)propyl)-4-
hydroxybutanamide
Theacrylic monomerwas synthesized usingtwosteps. In the first
step, a flame-dried, 2000-mL, three-neck, round-bottomed flask
was attached to a 250-mL addition funnel and a condenser. The
round-bottomed flask was charged with 50 mL (1.00 mol) of 3,3-
iminobis(N,N-dimethylpropylamine) and 700 mL tetrahydrofuran
(THF). The round-bottomed flask was purged with nitrogen. Diiso-
butylalumium hydride (1.0 M solution in toluene, 225 mL,1.00 mol)
was added to the addition funnel and subsequently added to the
reaction flask in a drop-wise fashion. The solution was stirred for 7 h
at 0 ꢁC. Subsequently, the reaction flask was warmed to room tem-
perature and 17 mL (1.00 mol) of g-butyrolactone was added to the
reaction mixture and the solution was refluxed for 12 h. Water
(10 mL) was added slowly to the cooled reaction flask and THF was
evaporated under reduced pressure. A 15% sodium hydroxide solu-
tion (100 mL) was added and stirred for an hour. The aqueous layer
was extracted three times with 100 mL dichloromethane and the
organic fractions were combined. The solvent was concentrated in
vacuo and the product was purified using Kugelrohr distillation. An
overall yield of 70% was obtained. 1H NMR (500 MHz, CDCl3): 1.71
(m, 4H, Hc),1.90 (m, 2H, Hf), 2.21 (d,12H, Ha), 2.26 (t, 4H, Hb), 2.52 (t,
2H, He), 3.35 (m, 4H, Hd), 3.67(t, 2H, Hg), 3.45 (s,1H, Hi). HRMS (ESþ):
m/z calcd for [M þ Hþ] 273.24 g/mol, found 274.17 g/mol (Fig. S1).
2. Experimental
2.1. Materials
3,3-Iminobis(N,N-dimethylpropylamine) (DMPA, 97%), acryloyl
chloride (97% ), 6-bromohexanoyl chloride (97%), and triethylamine
(TEA, 99%) were purchased from Aldrich and vacuum distilled prior
to use. Potassium tert-butoxide (99.99%), adenine (A, 99%), thymine
(T, 99%), poly(ethylene glycol) (PEG) with Mn ¼ 1000 g/mol, sodium
bicarbonate (99.7%), magnesium sulfate (99.5%), 1-bromobutane
(99%), and potassium carbonate (>99%) were obtained from
2.4. Synthesis of 4-(bis(3-(dimethylamino)propyl)amino)-4-
oxobutyl acrylate
SigmaeAldrich and used without further purification.
g-butyr-
olactone (þ99%) and diisobutylalumium hydride (1.0 M solution in
toluene) were purchased from Aldrich and used as received.
Dichloromethane (DCM, HPLC grade), tetrahydrofuran (THF, HPLC
grade), and dimethyl formamide (DMF, HPLC grade) were passed
through alumina and molecular sieve columns before use. Chloro-
form (CHCl3, HPLC grade) and ethyl acetate (EtOAc, HPLC grade)
were purchased from Fischer Scientific and used as received.
In the second step, a flame-dried, 100-mL, round-bottomed flask
was connected to a 50-mL addition funnel and was charged with
dichloromethane (DCM) and 1.00 eq of hydroxyl-containing diter-
tiary amine monomer that was synthesized from the first step. The
flask was cooled to 0 ꢁC and acryloyl chloride (1.20 eq) was added
to the addition funnel containing DCM and the solution was added
drop-wise to the reaction flask. The reaction was allowed to pro-
ceed for 12 h. Upon reaction completion, DCM was evaporated and
the salt was dissolved in a mixture of saturated NaHCO3 (aq) and
saturated Na2CO3 (aq). The aqueous solution was extracted six
times with DCM. The solution was concentrated in vacuo and the
product was purified using Kugelrohr distillation. A yellow oil with
an overall yield of 30% was obtained. 1H NMR (500 MHz, CDCl3):
1.68 (m, 4H, Hc), 2.02 (m, 2H, Hf), 2.19 (d, 12H, Ha), 2.24 (t, 4H, Hb),
2.42 (t, 2H, He), 3.31 (m, 4H, Hd), 4.20 (t, 2H, Hg), 5.78e5.83 (m, 1H,
HJ1), 6.05e6.14 (m, 1H, Hk), 6.35e6.42 (m, 1H, HJ2) (Fig. S2).
2.2. Instrumentation
1H NMR spectroscopic analyses were performed on Varian
Advance 500 MHz spectrometer to confirm monomer and polymer
compositions at ambient temperature in CDCl3 or CD3OD. Fast
Atom Bombardment Mass Spectrometry (FAB-MS) was conducted
in positive ion mode on a JEOL HX110 dual focusing mass spec-
trometer. Differential scanning calorimetry (DSC) was conducted
on a TA Instruments Q100 under a nitrogen flush of 50 mL/min at
a heating rate of 10 ꢁC/min. The glass transition temperatures were
measured as the midpoint of the transition in the second heating
scan. Thermogravimetric analysis (TGA) was conducted on a TA
Instruments Hi-Res TGA 2950 under nitrogen at a heating rate of
10 ꢁC/min. Atomic force microscopy (AFM) was performed using
a Veeco MultiMode (Veeco Instruments, Plainview, NYA). AFM was
2.5. Synthesis of bromine end-capped PEG (Br-PEG-Br)
The commercially available 1000 g/mol poly(ethylene glycol)
(PEG) (10 g, 1.00 mol) was introduced to a 250-mL, two-neck,
round-bottomed flask equipped with a magnetic stir bar, addition

1590
M. Tamami et al. / Polymer 54 (2013) 1588e1595
funnel, and nitrogen inlet. Anhydrous dichloromethane (100 mL)
was added to the round-bottomed flask and 50 mL was added to
the addition funnel. The flask was cooled to 0 ꢁC and 6-
bromohexanoyl chloride (2.20 eq) was added to the addition fun-
nel with a syringe and subsequently added to the reaction flask in
a drop-wise fashion. The reaction was allowed to proceed for 24 h.
Upon reaction completion, the reaction mixture was washed twice
with saturated NaHCO3 (aq) and twice with distilled water. The
DCM layer was separated and dried over magnesium sulfate. The
solution was concentrated in vacuo and dried under vacuum at
100 ꢁC for 12 h (97% yield). The 1H NMR number-average molecular
weight was 1300 g/mol. The 1H NMR (400 MHz, CDCl3) spectro-
scopy for the bromine end-capped 1K PEG is as follows:
tBuOK for 4 d. The nucleobase-containing ionene product was
precipitated in ethyl acetate and dried in vacuo (0.1 mmHg) for 24 h
(90% yield). 1H NMR (400 MHz, CD3OD) for adenine-containing
ionene: 1.42 (m, 4H, Hm), 1.70 (m, 4H, Hn), 1.81 (m, 4H, Hi), 1.91
0
(p, 2H, Hd ), 2.06 (m, 4H, Hd), 2.30 (t, 2H, He), 2.40 (m, 4H, Hp), 3.01
(t, 2H, Hh), 3.12 (d, 12H, Ha), 3.33e3.59 (m, 8H, Hbþcþg), 3.63 (s, 70H,
Hk1þk2), 3.69 (t, 4H, Hl), 4.15 (t, 2H, Hq), 4.20 (m, 4H, Hf), 4.52 (m, 2H,
Hj), 8.16 (s, 1H, Hs), 8.22 (s, 1H, Hr).1H NMR (400 MHz, CD3OD) for
thymine-containing ionene: 1.44 (m, 4H, Hm), 1.71(m, 4H, Hn), 1.81
0
(m, 4H, Hi), 1.86 (s, 3H, Hs), 1.95 (p, 2H, Hd ), 2.08 (m, 4H, Hd), 2.32(t,
2H, He), 2.40 (t, 4H, Hp), 2.77 (t, 2H, Hh), 3.13 (d, 12H, Ha), 3.34e3.59
(m, 8H, Hbþcþg), 3.63 (s, 70H, Hk1þk2), 3.69 (t, 4H, Hl), 4.00 (t, 2H,
Hq), 4.17 (t, 2H, Hj), 4.20 (t, 4H, Hf), 7.49 (s, 1H, Hr) (Fig. S7).
d
¼ 1.47 ppm (m, 4H, per chain, Hc), 1.65 ppm (m, 4H, Hd), 1.87 ppm
(m, 4H, Hb), 2.35 ppm (t, 4H, He), 3.40 ppm (t, 4H, Ha), 3.64 ppm (m,
76H, Hh), 3.69 ppm (m, 4H, Hg), 4.22 ppm (m, 4H, Hf) (Fig. S3).
2.10. Preparation of ionene blend with guest molecules
The segmented adenine-containing ionene and thymine-
containing ionene solutions in chloroform were mixed with nBT
and nBA chloroform solutions in a 1:1 molar ratio, respectively. The
blends were stirred for an hour, and cast in TeflonÒ molds.
Chloroform slowly evaporated at room temperature for 48 h and
the films were annealed at 100 ꢁC for 24 h in vacuo.
2.6. Synthesis of n-butyl thymine (nBT) guest molecule
Thymine (10.05 g, 80.0 mmol), potassium carbonate (11.06 g,
80.0 mmol), 1-bromobutane (3.67 g, 26.8 mmol), and DMSO
(200 mL) were added to a 500-mL round-bottomed flask. The so-
lution was heated to 50 ꢁC for 8 h and then cooled. The resulting
precipitate was removed through filtration and the DMSO solution
was added to 600 mL water. The aqueous solution was extracted
four times with 100 mL DCM and then the organic layer was washed
four times with 300 mL water. The organic layer was dried over
magnesium sulfate and then concentrated in vacuo to obtain a solid.
The solid product was recrystallized from chloroform:hexanes to
obtain a white solid with a yield of 51%. 1H NMR (400 MHz, CDCl3):
0.95 ppm (t, 3H, Ha), 1.36 ppm (m, 2H, Hb), 1.66 ppm (p, 2H, Hc),
1.92 ppm (s, 3H, He), 3.69 ppm (t, 2H, Hd), 6.97 ppm (s, 1H, Hf),
8.65 ppm (s, 1H, Hg) (Fig. S4).
3. Results and discussion
3.1. Synthesis of nucleobase-functionalized PEG-based ionene
homopolymers
Synthesis of nucleobase functional PEG-based ionenes involved
the synthesis of an acrylic ditertiary amine monomer and an oli-
gomeric bromine-terminated PEG spacer. In the first synthetic step
of the acrylic monomer synthesis, the secondary amine ring-
opened
g-butyrolactone in the presence of DIBAL, an efficient
amidating agent for the conversion of lactones to amides [22]. Thus,
the reaction produced an OH-containing ditertiary amine monomer
in high yields. In the second step, reaction between the primary
alcohol and acryloyl chloride yielded an acrylic ditertiary amine
monomer (Scheme 1a). Difunctional bromine-terminated PEG was
synthesized in a similar fashion to earlier (Scheme 1b) bromine
end-capped poly(propylene glycol) oligomers, and MALDI-TOF
mass spectroscopy and titration analysis confirmed their difunc-
tionalities [9].
As shown in Scheme 2, the pure, difunctional tertiary amine and
bromide monomers reacted according to Menshutkin reaction
conditions to provide an acrylate-containing PEG-based ionene.
Post-polymerization functionalization without isolation of the
precursor using base-catalyzed Michael addition to the acrylate
ionene precursor in DMF yielded the adenine-containing ionene
(ionene-A) and thymine-containing ionene (ionene-T). The Michael
addition reaction solution initially was heterogeneous and became
homogeneous upon reaction due to the enhanced solubility of the
final nucleobase ionene. The thermodynamically controlled, base-
catalyzed Michael addition promoted a regioselective substitution
of adenine and thymine at the N9 and N1 positions, respec-
tively.[23] Optimization of the reaction conditions, such as solvent,
temperature, and base, resulted in regioselective nucleobase-
containing ionenes. 1H NMR spectroscopy confirmed successful
incorporation of the heterocyclic bases, and disappearance of the
olefinic protons at 5.8e6.6 ppm confirmed quantitative addition of
the nucleobases to the acrylate ionene precursor.
2.7. Synthesis of n-butyl adenine (nBA) guest molecule
Adenine
(6.05 g,
44.8 mmol), 1-bromobutane
(6.16 g,
45.0 mmol), potassium carbonate (8.33 g, 60.3 mmol), and DMSO
(60 mL) were added to a 250-mL round-bottomed flask. The result-
ing solution was stirred for 48 h at 23 ꢁC and then poured into
600 mL water. The aqueous solution was extracted three times with
100 mLDCMandthentheorganiclayerwaswashed threetimes with
100 mL water. The organic layer was dried over magnesium sulfate
andconcentratedinvacuotoobtainasolid. Theproductwasobtained
as a white solid after recrystallization from chloroform:hexanes. 1H
NMR (400 MHz, CDCl3): 0.96 ppm (t, 3H, Ha), 1.37 ppm (m, 2H, Hb),
1.88 ppm (p, 2H, Hc), 4.20 ppm (t, 2H, Hd), 5.67 ppm (s, 2H, He),
7.79 ppm (s, 1H, Hf), 8.37 ppm (s, 1H, Hg) (Fig. S5).
2.8. Synthesis of acrylate-containing PEG-based ionene precursor
Upon the synthesis of acrylic ditertiary amine monomer and
bromine end-capped PEG, a 1:1 ratio of monomers was polymerized
in DMF for 24 h at 80 ꢁC in the presence of a catalytic amount of BHT.
The polymer was stored in the DMF solution until the next synthetic
step. 1H NMR (400 MHz, CD3OD):1.42 (m, 4H, Hm), 1.72 (m, 4H, Hn),
1.81 (m, 4H, Hi), 2.05 (m, 6H, Hd), 2.40 (m, 4H, Hp), 2.55 (t, 2H, He),
3.11 (d, 12H, Ha), 3.33e3.47 (m, 8H, Hbþc), 3.50 (t, 4H, Hg), 3.63 (s,
75H, Hk1þk2), 3.69 (t, 4H, Hl), 4.18e4.27 (m, 6H, Hf), 5.88e5.93 (dd,
1H, Hj2), 6.13e6.22 (m, 1H, Hh), 6.36e6.42 (dd, 1H, Hj1) (Fig. S6).
2.9. Synthesis of nucleobase-containing PEG-based ionene using
post-polymerization functionalization
3.2. 1H NMR titrations
After polymerization, the acrylic ionene solution in DMF was
charged with 1.20 mol of adenine or thymine and 0.30 mol of
When adenine and thymine nucleobases form complementary
hydrogen bonds, the chemical shift for the NH (thymine) and NH2

M. Tamami et al. / Polymer 54 (2013) 1588e1595
1591
a)
b)
Scheme 1. Synthesis of acrylic ditertiary amine monomer (a) and bromine end-capped 1000 g/mol PEG (b).
Scheme 2. Post-polymerization functionalization of PEG-based ionene.

1592
M. Tamami et al. / Polymer 54 (2013) 1588e1595
0
-2
y = -9.7465x - 0.1896
R² = 0.9968
-4
-6
-8
-10
-12
-14
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1/[UOP+] (mM-1)
Fig. 1. BenesieHildebrand plot of ionene-A and UOPþ guest molecule association in CDCl3.
(adenine) protons shift downfield relative to their original peak
position [24]. We blended a 1:1 molar ratio of [ionene-A]:[ionene-
T] at a 4 mM nucleobase in chloroform to investigate the formation
of complementary multiple hydrogen bonds. However, 1H NMR
resonances of NH and NH2 protons for the [ionene-A]:[ionene-T]
blend had insignificant change in their chemical shifts compared
to the ionene homopolymers. In order for the pendant nucleobases
to interact, both PEG-based ionene chains must be in close prox-
imity. Due to the charged nature of the ionene backbone and steric
hindrance between two bulky nucleobase-containing PEG chains,
the formation of a hydrogen bond between the complementary
nucleobases was presumably restricted. In order to examine the
influence of charge and bulkiness of complementary nucleobases,
uracil octyl phosphonium salt (UOPþ) served as a complementary,
low molar mass charged guest molecule for the [ionene-A]. For
each repeat unit, the charge ratio of [ionene-A] to [UOPþ] was 2:1.
Therefore, the charge density per repeat unit decreased, and the
bulkiness of the complementary nucleobase decreased with a low
molecular weight guest molecule compared to high molecular
weight PEG ionene. Solutions of [ionene-A]:[UOPþ] complexes
were prepared where the [ionene-A] concentration remained
constant at 4 mM and the [UOPþ] concentration systematically
increased. The position of the NH2 resonance of ionene-A in the
complex shifted downfield (from 6.27 to 6.64 ppm) with the
increase in [UOPþ] concentration. The association constant (Ka)
based on BenesieHildebrand plot (Fig. 1) was 19 Mꢀ1, which was in
acceptable range (10e100 Mꢀ1), however, the value was low com-
pared to the neutral guest molecules. Although chargeecharge
repulsion reduced the strength of association between comple-
mentary bases, less sterically hindered guest molecule promoted
hydrogen bonding interactions. Thus, we synthesized neutral
adenine and thymine-containing small guest molecules to examine
the supramolecular assembly of the low molar mass nucleobase
guests with the nucleobase ionenes. These guest molecules have
low molar mass (190 g/mol), without charge, and the guests are
highly soluble in chloroform, which makes them ideal candidates
for interaction with the complementary ionene homopolymers.
The stoichiometry of the host-guest complex was necessary
before calculating their association constant (Ka) [25]. Job’s analysis,
a continuous variation method, elucidated the host-guest stoichi-
ometry using 1H NMR spectroscopy [26,27] Solutions containing
host nucleobase ionenes and low molar mass nucleobase guests
were prepared in chloroform. The total solution concentration
maintained constant, while the molar ratios of the two components
varied. The adenine NH2 resonance shifted at different molar
fractions of [ionene-T]:[nBA], and [nBT]:[nBA] complexes and thy-
mine NH chemical shift at different molar fractions of [ionene-
A]:[nBT]. Fig. 2 demonstrates Job’s plots for [ionene-A]:[nBT],
(a)
0.15
[nBA]:[nBT]
0.12
0.09
0.06
0.03
0
0
0.2
0.4
0.6
X (nBT)
0.8
1
(b)
0.15
0.12
0.09
0.06
0.03
0
[ionene-T]:[nBA]
[ionene-A]:[nBT]
0
0.2
0.4
0.6
0.8
1
X (ionene)
Fig. 2. Job’s plot to determine the stoichiometry of (a) [nBT]:[nBA], (b) [ionene-
T]:[nBA], and [ionene-A]:[nBT] complexes in CDCl3.

M. Tamami et al. / Polymer 54 (2013) 1588e1595
1593
[ionene-T]:[nBA], and [nBT]:[nBA]. The x-axis value of the parabolic
(a)
maximum of the Job’s plot represents the stoichiometry of the
complex. All plots were symmetric and had a maximum at a 0.5
molar fraction, which confirmed that base pairing occurred in a 1:1
fashion. Therefore, a maximum concentration of the complexes
formed at a 1:1 equimolar concentration of host and guest [28].
1H NMR titration experiments in chloroform, which is a solvent
that favors hydrogen bonding interactions due to the relatively low
dielectric constant, determined the association constants (Ka) be-
tween the adenine and thymine nucleobases. Solutions of [ionene-
A]:[nBT] complexes were prepared where the [nBT] concentration
remained constant at 4 mM and the [ionene-A] concentration
systematically increased from 4 mM to 16 mM. The position of the
NH resonance of nBT in the complex shifted downfield (from 8.11 to
8.35 ppm) with an increase in [ionene-A] concentration. The cur-
vature of chemical shift data with increasing adenine concentration
remained consistent with typical 1H NMR titration curves [29]. The
change in chemical shift with complexation results from a faster
exchange between associated and dissociated A-T complex on the
NMR time scale [25,30].
0.5
0.4
0.3
0.2
0.1
0
0
5
10
15
20
[ionene-A] (mM)
(b)
0
-1
-2
-3
-4
-5
-6
y = -13.77x - 1.2898
R² = 0.9955
The BenesieHildebrande model is a mathematical method to
determine the association constant (Ka) from an NMR titration
experiment. This model fits the nonlinear chemical shift data for
a dimeric hydrogen bond association assuming the complex is
formed in 1:1 stoichiometry [25,31]. The association constant (Ka)
from the BenesieHildebrand analysis is calculated using the
equation: 1/Dd ¼ 1/(KaDdmax[ionene-A]) þ 1/Ddmax. The Ddmax is the
maximum change of the chemical shift of the thymine NH proton.
The slope of the double reciprocal plot is 1/KaDdmax and the inter-
cept is 1/Ddmax. Fig. 3 demonstrates a typical nonlinear NMR titra-
tion curve of induced chemical shift versus solution concentration.
Fitting of this data to the BenesieHildebrande method produced
a linear, double reciprocal plot based on the association of AeT
complex, which further confirmed the 1:1 stoichiometry (Fig. 3).
The Ka for the supramolecular assembly of nBT and ionene-A was
94 Mꢀ1, which was consistent with earlier reports on adenine-
thymine base pair recognition (10e100 Mꢀ1 in CDCl3) [28,32,33].
Similar 1H NMR titration experiments were suitable for the
[ionene-T]:[nBA] complex. The concentration of [nBA] was 4 mM,
and we systematically increased the [ionene-T] concentration from
4 mM to 16 mM. The position of the NH2 resonance of nBA in the
complex shifted downfield (from 5.61 to 5.68 ppm) with an
increase in [ionene-T] concentration. The linear fit to the Benesie
Hildebrand model also confirmed a 1:1 stoichiometry. Fig. 4 de-
picts the double reciprocal plot of BenesieHildebrand for the as-
sociation of [ionene-T] and [nBA]. The Ka calculated from the slope
0.00
0.05
0.10
0.15
0.20
0.25
0.30
1/[ionene-A] (mM-1)
Fig. 3. (a) Nonlinear relationship between induced change for thymine NH chemical
shift and ionene-A concentration, and (b) BenesieHildebrand plot of ionene-A and nBT
guest molecule association in CDCl3.
understand the association of nucleobase pairs. All films were so-
lution cast from chloroform and annealed at 100 ꢁC for 24 h in
vacuo. DSC thermograms for the nucleobase-functionalized
ionene homopolymers and complexes showed a single glass
transition temperature at approximately ꢀ40 ꢁC. This transition
(a)
0.16
0.12
0.08
0.04
0
of this plot is 130 Mꢀ1
.
For comparison, 1H NMR titrations were performed with the
nBA and nBT guest molecules. The association constant Ka based on
the slope of the plot represented in Fig. 5 was 137 Mꢀ1. Although
the Ka values calculated for the three complexes are quite compa-
rable and within acceptable range for adenine-thymine interaction,
the similarity of Ka values of 130 Mꢀ1 for [ionene-T]:[nBA] and
[nBA]:[nBT] with Ka of 137 Mꢀ1 was attributed to better solubility of
ionene-T compared to ionene-A in CDCl3. This presumably led to
efficient accessibility of nucleobases and stronger association be-
tween nucleobase pairs in solution.
0
5
10
15
20
[ionene-T] (mM)
(b)
0
-4
y = -37.323x - 4.8535
-8
-12
-16
3.3. Thermal transitions
0
0.05
0.1
0.15
0.2
0.25
0.3
Upon discovering the complementary behavior of nucleobase-
containing ionenes with guest molecules in solution, we inves-
tigated the solid state properties of 1:1 molar ratios of ionene
complexes with small guest molecules. We studied the thermal
properties of ionene-A, ionene-T, and their complexes to
1/[ionene-T] (mM-1)
Fig. 4. (a) Nonlinear relationship between induce change for adenine NH2 chemical
shift and ionene-T concentration, and (b) BenesieHildebrand plot of ionene-T and nBA
guest molecule association in CDCl3.

1594
M. Tamami et al. / Polymer 54 (2013) 1588e1595
Table 1
(a)0.4
Thermal transitions of nucleobase-containing ionenes and their blends.
Sample
Tg (ꢁC) DSC
Td5% (ꢁC) TGA
0.3
0.2
0.1
0
Ionene-A
Ionene-T
[Ionene-A]:[nBT]
[Ionene-T]:[nBA]
ꢀ36
ꢀ40
ꢀ31
ꢀ48
246
248
243
222
crystallization and melting peak of nBT and nBA were absent from
the DSC thermograms (Fig. 6), and films were optically clear. Pre-
viously Mather and Long et al. [21] also showed that the addition of
uracil-containing phosphonium salt to the adenine-containing
triblock copolymers resulted in the disappearance of the phos-
phonium salt melting peak in the DSC thermograms. Thus, the
absence of melting and crystallization peaks of the guest molecules
indicated a well-defined hydrogen bonding interaction between
the polymer and the guest molecule. Table 1 illustrates the thermal
transitions of ionene homopolymers and ionene complexes.
0
2
4
6
8
10
[nBT] (mM)
(b)
0
y = -10.025x - 1.3713
R² = 0.9926
-4
-8
-12
-16
3.4. Morphology
Atomic force microscopy (AFM) and X-ray scattering on
annealed films of ionenes and ionene complexes ascertained the
morphology. Fig. 7 includes AFM phase images of ionene-A and
a 1:1 complex with nBT. The ionene-A revealed a microphase-
separated morphology, the darker regions corresponded to the
PEG SS (78 wt%) and the lighter regions corresponded to the harder
ionic domains and heterocyclic nucleobases (22 wt%). Comparing
the ionene segmented homopolymers with the blends showed
a decreased phase contrast suggesting the disruption of the
adenine-adenine hard phase through incorporation of comple-
mentary guest molecule. Fig. 8 illustrates the corresponding SAXS
data. Due to the difference in electron density of the HS relative to
the SS, a single peak was observed in the SAXS profile of ionene-A.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1/[nBT] (mM-1)
Fig. 5. (a) Nonlinear relationship between induced change for adenine NH2 chemical
shift and nBT concentration, and (b) BenesieHildebrand plot of nBT and nBA guest
molecule association in CDCl3.
corresponded to the Tg of the PEG soft segment (SS) (1000 g/mol)
and confirmed a microphase separation of PEG SS from the ionic
hard segment (HS). Since the nucleobases were incorporated into
the hard phase, the hydrogen bonding interactions did not sig-
nificantly influence the Tg of the SS. In the solution cast 1:1 blend of
[ionene-A]:[nBT] and [ionene-T]:[nBA] from chloroform, the
0.40
20
15
10
5
[Ionene-A]:[nBT] = 1:1
[Ionene-A] = 100 mol%
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
[nBT]
0
-5
-80
-30
20
70
120
170
220
Temperature (°C)
Fig. 6. DSC thermograms of ionene-A homopolymer and 1:1 complex with nBT. Second heating cycle is shown.

M. Tamami et al. / Polymer 54 (2013) 1588e1595
1595
Fig. 7. AFM phase images of ionene-A (1) and 1:1 complex of [ionene-A]:[nBT] (2).
ionenes. AFM phase images showed a decrease in phase contrast
with the incorporation of complementary guest molecules, and the
SAXS revealed a broader peak at higher q values.
1000
100
10
[Ionene-A]
[Ionene-A]:[nBT]
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.polymer.2013.01.040.
1
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