Synthesis and characterization of polyphenylenes with polypeptide and poly(ethylene glycol) side chains

Article abstract of DOI:10.1002/pola.27621

We report a novel approach for fabrication of multifunctional conjugated polymers, namely poly(p-phenylene)s (PPPs) possessing polypeptide (poly-l-lysine, PLL) and hydrophilic poly(ethylene glycol) (PEG) side chains. The approach is comprised of the combination of Suzuki coupling and in situ N-carboxyanhydride (NCA) ring-opening polymerization (ROP) processes. First, polypeptide macromonomer was prepared by ROP of the corresponding NCA precursor using (2,5-dibromophenyl)methanamine as an initiator. Suzuki coupling reaction of the obtained polypeptide and PEG macromonomers both having dibromobenzene end functionality using 1,4-benzenediboronic acid as the coupling partner in the presence of palladium catalyst gave the desired polymer. A different sequence of the same procedure was also employed to yield polymer with essentially identical structure. In the reverse sequence mode, low molar mass monomer (2,5-dibromophenyl)methanamine, and PEG macromonomer were coupled with 1,4-benzenediboronic acid in a similar way followed by ROP of the L-Lysine NCA precursor through the primary amino groups of the resulting polyphenylene.

Full text of DOI:10.1002/pola.27621

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Synthesis and Characterization of Polyphenylenes with Polypeptide and
Poly(ethylene glycol) Side Chains
Huseyin Akbulut,1 Takeshi Endo,2 Shuhei Yamada,2 Yusuf Yagci1,3
1Department of Chemistry, Istanbul Technical University, Istanbul 34469, Turkey
2Molecular Engineering Institute, Kinki University, 11-6 Kayanomori, Iizuka, Fukuoka 820–8555, Japan
3Center of Excellence for Advanced Materials Research (CEAMR) and Chemistry Department, Faculty of Science, King Abdulaziz
University, P.O. Box 80203, Jeddah, 21589, Saudi Arabia
Correspondence to: Y. Yagci (E-mail: yusuf@itu.edu.tr)
Received 22 December 2014; accepted 19 February 2015; published online 28 March 2015
DOI: 10.1002/pola.27621
ABSTRACT: We report a novel approach for fabrication of multi-
functional conjugated polymers, namely poly(p-phenylene)s
(PPPs) possessing polypeptide (poly-L-lysine, PLL) and hydro-
philic poly(ethylene glycol) (PEG) side chains. The approach is
comprised of the combination of Suzuki coupling and in situ
N-carboxyanhydride (NCA) ring-opening polymerization (ROP)
processes. First, polypeptide macromonomer was prepared by
ROP of the corresponding NCA precursor using (2,5-dibromo-
phenyl)methanamine as an initiator. Suzuki coupling reaction
of the obtained polypeptide and PEG macromonomers both
having dibromobenzene end functionality using 1,4-benzenedi-
boronic acid as the coupling partner in the presence of palla-
dium catalyst gave the desired polymer. A different sequence
of the same procedure was also employed to yield polymer
with essentially identical structure. In the reverse sequence
mode, low molar mass monomer (2,5-dibromophenyl)me-
thanamine, and PEG macromonomer were coupled with 1,4-
benzenediboronic acid in a similar way followed by ROP of
the L-Lysine NCA precursor through the primary amino
C
V
groups of the resulting polyphenylene.
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KEYWORDS: conjugated polymer; N-carboxyanhydride; poly-L-
lysine; poly(ethylene glycol); poly(p-phenylene)s; polypeptide;
ring-opening polymerization; Suzuki coupling; biopolymers;
peptides; water-soluble polymers
advantages for CPs to be used in sensor technologies includ-
ing pH sensors, temperature sensors and recently developed
biosensors.27–29 In CPs, the adjusting molecular morphology
can lead to high surface area and extensive stability of the
enzymes incorporated. Variations at surface properties
depend on types of pendent groups, allowing for the produc-
tion of polymers which are water soluble or exceptionally
compatible with biomolecules.30,31
INTRODUCTION The extensive role of conjugated polymers
(CPs) in emerging synthetic bio-applications,1,2 optical,3 elec-
tronic,4–6 diodes,7,8 and display technologies,9 represents the
promising way in which considerable endeavors have been
directed towards the realization of many aspects of conduct-
ing organic polymers. The conducting properties of CPs arise
from having delocalized p-electron bonding along the poly-
mer backbone bearing a framework of alternating double
and single carbon-carbon bonds along which electrons can
flow.1,10–14 The mechanical flexibility, tunable optical proper-
ties, excellent conductivity, high mechanical strengths and
processability of some conducting polymers make them
potentially useful materials for new applications such as
high-contrast organic light-emitting displays and low-cost
organic photovoltaic solar cells.15–21 Conjugated structure of
CPs can create excellent one-dimensional surface for energy
transport of electrons and strong UV absorption.22–26 Espe-
cially, their fluorescence feature is one of the most suscepti-
ble to environmental change and this allows for eminent
selectivity in signaling reporter group materials and provides
Among vast number of CPs, poly(p-pheneylene)s (PPP)s are
the most promising class of polymer in terms of relatively
high photoluminescence, electroluminescence quantum effi-
ciencies and thermoxidative stability.32–34 PPPs can easily be
functionalized to give a wide range of derivatives due to the
fact that their syntheses generally include phenyl compound
monomers readily allowing for many chemical modifications.
Unfortunately, PPPs suffer from insolubility in many solvents,
which limit their processability.35–37 However, the addition of
conformational alkyl or water soluble pendants or polymers
as side chains to the backbone can lead to the obtainment of
Additional Supporting Information may be found in the online version of this article.
C
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soluble PPPs with high molecular weight. In related studies
in the authors’ laboratory, it has been shown that PPPs can
be combined with conventional synthetic polymers through
the combination of various chain polymerizations with cou-
pling processes to give soluble graft copolymers while pre-
serving the conjugated backbone properties.31,38,39
by the combination of Suzuki coupling and in situ NCA-ROP
processes. This amine compound was used as initiator for
NCA polymerization in the synthesis of polypeptide macro-
monomers and also used as monomer for the synthesis of
PPP copolymer bearing primary amine groups and PEG. We
provide two strategies to obtain PPP polymers bearing PEG
and poly-L-lysine (PLL) side chains; (1) Suzuki coupling poly-
merization between PEG and polypeptide macromonomers,
(2) NCA graft polymerization over the PPP backbone bearing
the primary amine and PEG groups. To investigate the struc-
ture of the polymers and monomers, 1H-NMR was applied.
UV-Vis and fluorescence spectra of PPP polymers are
accepted as evidence of the formation of p-conjugated back-
bone. The polymers obtained with the characteristic features
they possess could in the future be applied to various bio
mimicking and other bio applications.
Innovative approaches including the combination of polypep-
tides with polymeric structures have received enormous inter-
est in the fields of biomedicine,40 drug delivery,41,42 tissue
engineering,43 and they are regarded as impressive biomaterial
mimicking natural proteins.44 Such conjugation systems can
also open new perspectives for the fabrication of novel bioma-
terials that possess antibody/antigen, biocatalyst activity, com-
patibility with blood and/or tissue properties.45–47
Polypeptides, existing in three-dimensional conformations such
as a-helix, b-sheet, and random coil shapes, exhibit intriguing
morphological surface and excellent properties, such as low
toxicity, biodegradability, and biocompatibility.48,49 Recent
novel studies involving the synthesis of polymers such as poly-
ether, polyester, chitosan, polysiloxane etc., combined with
polypeptide segments, have the potential to revolutionize
today’s morphological structures and biomedical applications.50
Considering conventional methods which have some draw-
backs such as, multistep reactions, the use of hazardous
reagents such as phosgene, lack of sufficient monomer purity
and sensitivity to water contamination or heating conditions,
the ideal synthetic pathway for polypeptides is the ring-
opening polymerization (ROP) of the corresponding a-amino
acids of N-carboxyanhydrides (NCAs).51–57 In this technique,
the polymerization can readily be induced with primary or sec-
ondary amines and their living nature allows the decisive con-
trol of molecular weight and terminal structure. The precursor
urethane derivatives as monomer are readily synthesized by a
two-step procedure: (i) transformation of a-amino acids into
the corresponding ammonium salts and (ii) N-carbamylation of
the salts with diphenyl carbonates. These urethane derivatives
of a-amino acids are stable for several months at room tem-
perature.51 It should be noted that high stability and ease of
polymerization of these molecules with amines provides the
possibility to form polypeptide-polythiophene copolymers as
bio-based covering materials and ethanol biosensors.39
EXPERIMENTAL
Materials
Tetrakis (triphenylphosphine) palladium(0) (Pd(PPh3)4),
poly(ethylene glycol) mono methyl ether (Mn,RI 5 550 g
mol21) (Me-PEG550), N-boc-L-lysine, tetrabutylammonium
hydroxide (37% in methanol), 2,5-dibromo-benzoic acid,
N,N-dimethylacetamide (DMAc) 1,4-benzene-diboronic acid,
N-bromosuccinimide (NBS), 4-(N,N’-dimethyl) amino pyridine
(DMAP), dicyclohexylcarbodiimide (DCC), poly(ethylene gly-
col) mono methyl ether (PEG2000), Phthalimide potassium
salt, hydrazine monohydrate (98%), and benzoyl peroxide
are from Sigma-Aldrich and used without any further purifi-
cation. All solvents were purified and dried.
Measurements
1H-NMR spectra were performed with an Agilent VNMRS
500 MHz, and chemical shifts were recorded in ppm units
using tetramethylsilane as an internal standard. UV/Vis
absorbance measurements were recorded on a Shimadzu UV-
1601 spectrometer. Fluorescence measurements were per-
formed with a model LS-50 spectrometer from PerkinElmer
at room temperature. The fluorescence quantum yields were
determined according to IUPAC technical report using cou-
marine 1 (7-diethylamino-4-methylcoumarin) as standard.70
Gel permeation chromatography (GPC) measurements were
carried out in Viscotek GPCmax auto sampler system. Instru-
ment was equipped with a pump (GPCmax, Viscotek Corp.,
Houston, TX), light-scattering detector (k0 5 670 nm, Model
270 dual detector, Viscotek Corp.) consisting of two scatter-
ing angles: 7ꢀ and 90ꢀ and the refractive (RI) index detector
(VE 3580, Viscotek Corp.). Both detectors were calibrated
with PS standards in the narrow molecular weight distribu-
tion. Three ViscoGEL GPC columns (G2000HHR, G3000HHR
and G4000HHR) employed with THF in the 1.0 mL min21
flow rate at 30 ꢀC. All data were analyzed using Viscotek
OmniSEC Omni-01 software.
Although polymers bearing bound polypeptides provide
unique properties and advantage as regards biofunctionali-
zation, the use of soluble support is a more essential
requirement in biological application.58–63 Water solubility
can be achieved by the incorporation of polyethers, such as
poly(ethylene glycol) (PEG). Polypeptide-PEG combinations
as well as glycopeptides64–67 with various topological struc-
tures receive great attention due to their excellent solubility
in both water and organic solvents, and their biocompatibil-
ity. Moreover, they exhibit no antigenicity, immunogenicity,
or toxicity towards the human body and is approved by the
US American Food and Drug Administration (FDA).68,69
Synthesis of 1,4-Dibromo-2-(bromomethyl) benzene (1)
2,5-Dibromotoluene (2.55 mL, 18.5 mmol), NBS (6.2 g, 24.8
mmol) and 0.1 g of benzoyl peroxide were added to dry
Herein, we report a novel approach for the synthesis of PPP
copolymers bearing both PEG units and polypeptide groups
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40 mL of CCl4 under nitrogen atmosphere. The solution was
refluxed with condenser under nitrogen and 200 W light for
4 h. Heating started from 65 ꢀC and temperature was
ꢀ
increased by 10 C per hour and in the last 1 h temperature
ꢀ
was kept at 95 C. After that, the precipitate was filtered and
washed with a supplementary amount of CCl4 and finally
with a small quantity of CH2Cl2. The organic layer was
washed with water several times, and then dried over
NaSO4. The solvent was evaporated in vacuo. Then the
remaining crude material was purified by flash column chro-
matography (SiO2, Et2O) and recrystallized in petroleum
ether to yield (1) (3.04 g, 50%, white crystal). 1H-NMR
(CDCl3, 500 MHz): d 4.54 (s, 2H), 7.30 (dd, J 5 8.5, 2.4 Hz,
1H), 7.45 (d, J 5 8.5 Hz, 1H), 7.60 (d, J 5 2.3 Hz, 1H).
Synthesis of 2-(2,5-Dibromobenzyl)isoindoline-1,3-dione
(2)
To the solution of (1) (2.9 g, 8.8 mmol) in 10 mL dry DMF,
potassium phthalimide (2.45 g, 13.22 mmol) were added.
After stirring for 12 h at 160 ꢀC in a schlenck tube under
nitrogen atmosphere, the reaction mixture was poured into
water. The precipitate was washed with water, purified by
flash column chromatography (SiO2, CH2Cl2), precipitated in
ethanol and dried under vacuum to yield (2) (2.56 g, 74%,
SCHEME 1 Synthesis of MA-DBB via Gabriel reaction.
pressure. The crude products were purified by flash column
chromatography (SiO2, chloroform:acetone:acetic acid-
5 7:3:0.1, as eluent), and then recrystallized with ethyl ace-
tate/n-hexane to yield 3.4 g (68%), as white solids. 1H NMR
(500 MHz, CDCl3): d 1.34–1.58 (m, 4H), 1.67–1.83 (m, 1H),
1.84–1.96 (m, 1H), 3.15 (m, 2H), 4.39 (m, 1H), 4.86(s, 1H),
5.08 (s, 2H), 5.84 (d, 1H, J 5 8.1 Hz), 7.09 (d, 2H, J 57.8 Hz),
7.15 (t, 1H, J 57.4 Hz), 7.28–7.37 (m, 7H).
1
white solid). H-NMR (CDCl3, 500 MHz): d 4.94 (s, 2H), 7.26
(dd, J 5 5.7, 2.5 Hz, 1H), 7.28 (d, J 5 2.3 Hz, 1H), 7.45 (d,
J 5 8.3 Hz, 1H), 7.79 (dd, J 5 5.5, 3.0 Hz, 2H), 7.92 (dd,
J 5 5.5, 3.1 Hz, 2H).
Synthesis of (2,5-Dibromophenyl) Methane Amine (MA-
DBB)
Synthesis of Polypeptide Macromonomer (DBB-PLL)
To the solution of NE-carbobenzoxy-L-lysine (0.4 mg in 1 mL
DMAc) in a flame-dried Schlenk tube, the amine initiator,
MA-DBB, (5.3 mg in 0.5 mL DMAc) was added. The reaction
mixture was stirred at 60 ꢀC for 120 h under argon atmos-
phere. After the polymerization, mixture was cooled to room
temperature and poured into diethyl ether. The precipitates
were filtrated, and then dried under vacuum to yield 230 mg
(80%), as white solids. 1H-NMR (CDCl3, 500 MHz): d 1.29–
1.77 (broad), 1.78–2.01 (broad), 2.93–3.27 (broad), 3.49 (dd,
J 5 14.0, 7.0 Hz, 2H), 3.78–3.98 (broad), 4.85–5.25 (broad),
5.32–5.64 (broad), 6.84 (d, J 5 7.9 Hz, 1H), 7.06 (s, 1H),
7.17–7.36 (broad), 7.39 (d, J 5 8.5 Hz, 1H).
The solution of (2) (0.381 g, 0.95 mmol) in 25 mL absolute
ethanol with 3 mL hydrazine were stirred at 90 ꢀC for 5 h.
After allowing to cool to room temperature, the mixture was
extracted with CH2Cl2 and 10% NaHCO3. The organic layer
was dried over Na2SO4 and reduced in vacuo. The residue
was purified by column chromatography (Al2O3 (neutral),
1
THF) to yield (3) (145 mg, 57%, yellow oil). H-NMR (CDCl3,
500 MHz): d 1.59 (br, 2H), 3.87 (s, 2H), 7.23 (dd, J 5 8.4, 2.4
Hz, 1H), 7.38 (d, J 5 8.4 Hz, 1H), 7.54 (d, J 5 2.4 Hz, 1H).
Synthesis of Urethane Derivatives of NE-Carbobenzoxy-L-
Lysine (NCA-LL)
To a stirred solution of NE-carbobenzoxy-L-lysine (3.5 g, 12.5
mmol) in methanol (35 mL) and distilled water (5 mL), tet-
rabutylammonium hydroxide (37% in methanol) (8.77 g,
12.5 mmol) was slowly added at room temperature. After
stirring for 1 h, the mixture was concentrated under reduced
pressure. The residues were dissolved in acetonitrile
(25 mL). The solution was added dropwise over 10 min to a
stirred solution of DPC (2.68 g, 12.5 mmol) in acetonitrile
(25 mL) at room temperature, and then the mixture was
stirred overnight. To the reaction mixture, 1N of HCl aqueous
solution (20 mL) was added. The mixture was put into a
separating funnel containing distilled water (30 mL) and
ethyl acetate (30 mL). The aqueous layer was extracted with
ethyl acetate (3 3 30 mL) and the organic layers were com-
bined, dried over Na2SO4 and concentrated under reduced
Synthesis of PEG Macromonomer (DBB-PEG)
2,5-Dibromo-benzoic acid (4 g, 14.3 mmol), DMAP
(174.7 mg, 1.43 mmol), Me-PEG2000-OH (25.74 g, 12.87
mmol) were dissolved in 80 mL of dry CH2Cl2 under nitro-
gen atmosphere. To this solution was added DCC (3.246 g,
15.7 mmol) in 20 mL dry CH2Cl2 drop-wise under nitrogen.
Then, the reaction mixture was stirred for 5 days at room
temperature. After that, the mixture was filtered and washed
with 250 mL of CH2Cl2. The organic phase was quenched
with 10% NaHCO3 and brine, and then dried over Na2SO4.
The solution was concentrated and passed through a silica-
gel column using CH2Cl2 as eluent. After removal of solvent
in vacuo, the polymer was precipitated in cold diethyl ether
1
to yield 26 g (95%). H-NMR (CDCl3, 500 MHz): d 3.85–3.46
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DBB (0.8 g,
3
mmol), DBB-PEG (2.3 g), benzene-1,4-
diboronic acid (0.663 g, 4 mmol), Pd(PPh3)4) (0.092 gꢀ, 0.08
mmol) was dissolved under nitrogen and stirred at 70 C for
4 days in the Schlenk tube under vacuum. After stirring, the
reaction mixture extracted with CH2Cl2 and small amount of
water. Organic phase was concentrated and precipitated in
excess diethyl ether to yield 1.85 g, white powder. 1H-NMR
(CDCl3, 500 MHz): d 2.06 (broad), 3.38 (broad), 3.45–3.8
(broad), 7.92–8.20 (broad), 7.6–7.9 (broad), 7.2–7.6 (broad).
Synthesis of PPP Conjugated Polymer Bearing Poly–L-
Lysine and PEG (PPP-g-PEG-PLL) by Route 2
PPP-NH2-g-PEG (0.9 g) was charged into flame-dried Schlenk
tube and then dissolved in 1 mL of DMAc, followed by addi-
tion of NCA precursor (NCA-LL)/DMAc solution (0.3 g/1
mL). The reaction mixture was stirred at 60 ꢀC for 48 h
under argon atmosphere. After the NCA ROP polymerization,
the mixture was cooled to room temperature and then pre-
cipitated in cold diethyl ether. The resulting precipitates
were filtered and dried under vacuum to yield 0.523 g
FIGURE 1 1H-NMR spectra of MA-DBB before (bottom) and
after (top) proton exchange.
1
(95%). H-NMR (CDCl3, 500 MHz): d 1.07–1.66 (road), 1.75–
(broad), 3.38 (broad), 7.45 (dd, J 5 8.5, 2.4 Hz, 1H), 7.52 (d,
J 5 8.5 Hz, 1H), 7.93 (d, J 5 2.4 Hz, 1H).
2.15 (broad), 2.91–3.20 (broad), 3.55 (t, 3H), 3.56–3.68
(broad), 3.77–3.72 (m, 2H), 3.81–4.07 (broad), 4.18–4.37
(broad), 4.91–5.11 (broad), 5.43–5.60 (broad), 7.08–7.36
(broad), 7.34–8.39 (broad).
Synthesis of PPP Conjugated Polymer Bearing Poly-L-
Lysine and PEG (PPP-g-PEG-PLL)
DBB-PLL (0.18 g), DBB-PEG (0.8 g), and benzene-1,4-
diboronic acid (74.58 mg, 0.45 mmol) was dissolved in a
solution of 2 mL of 2.0 M K2CO3 (aq.) and 3 mL of THF in a
Schlenk tube. The mixture was bubbled with nitrogen over a
period of 30 min and then, Pd(PPh3)4 (26 mg, 0.226 mmol)
was added. The reaction mixture was stirred at 70 ꢀC for 4
days in the absence of oxygen and light. After polymeriza-
tion, the reaction mixture was extracted with water and
CH2Cl2. Organic phase was collected and dried over Na2SO4.
After the concentration of mixture under reduced pressure,
the final polymer was precipitated in cold diethyl ether and
RESULTS AND DISCUSSION
The crucial part of the approach for the synthesis of PPPs
with macromolecular side chains is to obtain dibromoben-
zene functionalized polymers or low molar mass related
components since applied Suzuki coupling reactions utilize
dibromobenzenes in conjunction with the benzene diboronic
acids and derivatives. Thus, primary amino functional dibro-
mobenzene possessing suitable functionalities for both poly-
peptide incorporation and Suzuki coupling processes was
synthesized via Gabriel reaction.71–76 2,5-Dibromotoluene
was chosen as starting material because it can readily react
with N-bromosuccinimide (NBS) to afford the allylic bromi-
nation of the methyl group in the presence of peroxide. After
obtaining 1 in good yield, the substitution reaction with the
conjugate base of phthalimide gave the corresponding N-
alkyl phthalimide, 2-(2,5-dibromobenzyl)isoindoline-1,3-
dione, (2). The efficiency of the reaction was increased by
using preformed potassium salt of phthalimide, instead of
phthalimide, facilitating the reaction to be conducted in a
polar protic solvent such as DMF under homogenous condi-
tions. Treatment of the imide derivative, 2 with hydrazine in
methanol results in the formation of phthalhydrazide and
(2,5-dibromophenyl) methanamine (MA-DBB) through
1
dried under vacuum to yield 0.556 g (55%). H-NMR (CDCl3,
500 MHz): d 1.01–1.68 (broad), 1.69–2.09 (broad), 2.97–3.24
(broad), 3.40 (d, J 5 15.2 Hz, 3H), 3.41–3.71 (broad), 3.81–
3.76 (m, 2H), 3.82-3.92 (broad), 4.19–4.36 (broad), 4.93–
5.12 (broad), 5.37–5.52 (broad), 7.12–7.36 (broad), 7.35–
8.36 (broad).
Synthesis of PPP Conjugated Polymer Bearing Primary
Amine and PEG (PPP-NH2-g-PEG)
The reaction solution was prepared with 30 mL THF and
20 mL of aqueous K2CO3 solution under nitrogen. Prior use,
this solution was degassed by bubbling nitrogen over a
period of 30 min. In the 10 mL of the reaction solution, MA-
SCHEME 2 Synthetic route for the urethane derivative of a-amino acid, NCA-LL.
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SCHEME 3 Synthesis of DBB-PLL by in situ NCA ring opening polymerization.
intramolecular substitution. By using column chromatogra-
phy, the desired amine compound MA-DBB could be sepa-
rated from the side product phthalhydrazide. The overall
synthetic pathway is outlined in Scheme 1. The structure of
MA-DBB was confirmed by 1H-NMR analysis (Fig. 1). Singlet
peak at 1.59 ppm was assigned to ANH2 protons, which was
further confirmed by the examination of the hydrogen–deu-
terium exchange reaction. After the deuterium exchange
using D2O, the peak at 1.59 ppm disappeared and a new sin-
glet peak at 4.72 ppm appeared.
ROP were combined in two different sequences. In the first
route, polypeptide macromonomer (DBB-PLL) was prepared
by ROP of NCA-LL using MA-DBB as an initiator (Scheme 3).
In earlier studies, we have shown that at high amine concen-
tration, the monomer is rapidly consumed and polymeriza-
tion proceeds in
a
controlled manner.51 In the
polymerization process, the NCA ring is formed by the elimi-
nation of phenol. Then, the primary amine group of the ini-
tiator attacks to the carbonyl carbon of NCA ring to form an
amide bond between initiator and monomer with the evolu-
tion of CO2, and the other activated amine side of the opened
NCA ring ensues polypeptide chain growth.
As stated previously, polypeptides can be synthesized from
the corresponding NCA precursors. For our convenience, we
have selected L-lysine based NCA precursor, L-lysine-N (phe-
nyloxycarbonyl) amino acid (NCA-LL). This urethane deriva-
tives of a-amino acid, was synthesized in two reaction steps;
(i) the transformation of a-amino acid into the corresponding
ammonium salt by an ionic exchange reaction and (ii) N-car-
bamylation of the ammonium salt-protected amino acid via
nucleophilic attack of the active primary amino group to the
carbonyl group of diphenyl carbonate (Scheme 2).
The structure of the polypeptide and presence of the termi-
1
nal dibromobenzene group were confirmed by H-NMR anal-
ysis. As can be seen from Figure 2, aromatic protons of the
end group resonate at 7.47, 7.06, and 6.84 ppm.
Characteristic aromatic protons corresponding to the repeat-
ing unit appear between 7.13 and 7.42 ppm.
The other polymeric coupling component, PEG macromono-
mer was synthesized by the well-known Steglich esterifica-
tion method under mild conditions with the favorable
catalytic action of 4-dimethylaminopyridine (DMAP)31
The final product NCA-LL is stable and can be stored for sev-
eral months at room temperature. Starting material of N-
(phenoxycarbonyl) amino acid was synthesized according to
the described procedure.
1
(Scheme 4). H-NMR spectral analysis of DBB-PEG evidenced
the presence of both PEG repeating units and end group
(Fig. S1, Supporting Information).
Synthesis of PPPs with Polypeptide and PEG Side Chains
In order to develop the protocol for the synthesis of the final
polypeptide bioconjugate, Suzuki coupling, and in situ NCA
The assembling of PEG and polypeptide side chains on the
PPP backbone was achieved by Suzuki condensation poly-
merization that provides molecular design flexibility. In this
connection, it should be pointed out that Yamamoto cou-
pling of only macromonomers without any spacer units is
expected to yield much lower chain growth due to the
steric hindrance of the bulky polymer chains. In the applied
strategy, hydrophilic and biorelated segments can be incor-
porated to the PPP structure through the sequence adjust-
ment using
a spacer benzene ring. In the Suzuki
polymerization, macromonomers and the coupling partner
1,4-benzenediboronic acid in homogeneous suspension of
FIGURE 2 1H-NMR spectrum of polypeptide macromonomer,
SCHEME 4 Synthesis of PEG macromonomer via Steglich
DBB-PLL.
esterification reaction.
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SCHEME 5 Synthesis of PPP polymer bearing poly-L-lysine and
PEG side chains.
FIGURE 4 UV absorption spectra of DBB-PEG, DBB-PLL, and
PPP-g-PEG-PLL in DMF solution (0.1 mg mL21). [Color figure
can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
THF/water with potassium carbonate were reacted in the
presence of palladium catalyst (Scheme 5). The polymeriza-
tion time was deliberately prolonged to 4 days to overcome
steric limitations of the macromonomers and obtain graft
copolymers with adequate molecular weight. As polypep-
tides have tendency to form helical structures affecting the
solubility properties, the molar ratio of PEG macromonomer
to polypeptide macromonomer was deliberately kept low
(mol/mol; 6/1) so as to provide sufficient solubility (Sup-
porting Information, Table S1). 1H-NMR spectrum of the
resulting graft copolymer, PPP-g-PEG-PLL, presented in Fig-
ure 3, clearly verifies the presence of both PPP backbone
and side chains. Conjugated phenyl protons of PPP back-
bone appear as broad signals at 8.3 to 7.34 ppm. While
phenyl protons of PLL segment are detected at 7.32 and
7.16, the broad signals at 3.74 and 3.42 are attributed to
the CH2 protons of PEG side chain.
Expected strong absorption band of PPP-g-PEG-PLL was due
to the presence of supplementary conjugated phenylene
rings in the main chain.
Figure 5 shows the fluorescence spectra of PPP-g-PEG-PLL.
The influence of starting macromonomers was very low in
the observed spectrum of the graft copolymer, as their
independent fluorescence intensities are rather weak and
a nearly mirror-image-like relation between absorption
and emission spectra was observed. It should also be
noted that the fluorescence spectrum has a small shoulder
emission at lower wavelength which may arise from the
random placement of the macromonomer sequences in the
backbone. Moreover, the aggregations that are taking place
during the nanoparticles formation through self-
assembling of PPP backbones in solution create the red
shifted shoulders in asymmetric shape.77,78 In the second
Photophysical characteristics of the graft copolymer were
also investigated. UV-Vis absorption of spectra of PPP-g-PEG-
PLL and its precursors, PEG and PLL macromonomers regis-
tered in DMF with the same concentration are presented in
Figure 4. As can be seen, DBB-PLL macromonomer has rela-
tively stronger absorption than that of the DBB-PEG macro-
monomer due to the aromatic phenyl groups in the
structure.
FIGURE 5 Fluorescence excitation and emission spectra of
PPP-g-PEG-PLL in DMF (1.43 mg mL21). [Color figure can be
viewed in the online issue, which is available at wileyonlineli-
brary.com.]
FIGURE 3 1H-NMR spectrum of PPP-g-PEG-PLL in CDCl3.
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SCHEME 6 The synthesis of PPP-NH2-g-PEG by Suzuki cou-
pling polymerization.
SCHEME 7 The synthesis of PPP-g-PEG-PLL by NCA ROP of
NCA-LL by using PPP-NH2-g-PEG.
route, the sequence of the processes of the already
described strategy was reversed. The usual Suzuki cou-
pling reaction described in the first route using MA-DBB,
DBB-PEG, and p-benzenediboronic acid under similar
experimental conditions yielded PPP with amino groups in
the structure (Scheme 6).
tophysical properties of the polymers investigated in dilute
DMF and water solutions are depicted in Table S2 (Support-
ing Information).
In the process, the mole ratio of MA-DBB to DBB-PEG was
3/1 so as to obtain polymers with longer chain length. As
the relative amount of DBB-PEG is increased, due to its poly-
meric nature and steric hindrance, limited chain growth is
expected.
In the final step for the synthesis of PPP bearing PEG and
PLL side chains, NCA ROP of NCA-LL was conducted through
the primary amino groups of PPP-NH2-g-PEG exactly under
the same polymerization conditions described in the first
route. The overall reaction is depicted in Scheme 7. The 1H-
NMR spectrum of the graft copolymer exhibited almost the
same spectral characteristics to that obtained by the first
route since both methods yield essentially structurally iden-
tical polymers.
The structure of the amino-functional PEG grafted PPP (PPP-
1
NH2-g-PEG) was confirmed by H-NMR analysis and Figure 6
exhibits the typical proton signals of PEG and PPP units as
well as that of the primary amine at 1.96 ppm. Notably, the
latter protons disappear after D2O proton exchange reaction.
Compared with the precursors, PPP-NH2-g-PEG shows strong
UV absorbance and fluorescence emission at higher wave-
lengths due to the extended conjugation arising from the
PPP structure (Supporting Information, Figs. S2 and S3) Pho-
However, the aromatic protons corresponding to the PPP
backbone were broader and more intense (Supplementary
Information, Fig. S4). This is expected since the Suzuki cou-
pling reaction was carried out with low molar mass mono-
mer, MA-DBB, facilitates longer PPA chain growth. This was
further confirmed by the molecular weight measurements.
As can be seen from Table 1, where molecular weight char-
acteristics of the graft copolymers obtained by the two
routes and intermediate polymers formed at various stages
are compiled.
TABLE 1 Molecular Weight Characteristics of the Graft Copoly-
mers and Precursors
Mw (g mol21
)
Mw/Mn
DPa
a
a
Polymer
DBB-PLL
86,660
6,880
2.01
1.24
1.85
1.41
2.41
328
150
–
DBB-PEG
PPP-g-PEG-PLL (route 1)
PPP-NH2-g-PEG
PPP-g-PEG-PLL (route 2)
140,450
34,430
41,810
–
–
a
Weight-average molecular weight (Mw), molecular weight distribution
(Mw/Mn), and degree of polymerization (DP) were determined by GPC
with light scattering detector according to polystyrene standards.
FIGURE 6 1H-NMR spectra of PPP-NH2-g-PEG before (bottom)
and after (top) proton exchange.
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12 S. W. Thomas, T. M. Swager, Macromolecules 2005, 38,
CONCLUSIONS
2716–2721.
In conclusion, in this work we presented a design principle
to fabricate conjugated PPP graft copolymers with hydro-
philic PEG and polypeptide side chains by the combination
of NCA ROP and Suzuki polycondensation processes via two
routes. In the first route, the dibromo benzene functional
PEG and PLL macromonomer obtained by in situ NCA ROP
were used in a Suzuki coupling reaction using benzene
diboronic acid as the antagonist Suzuki coupling agent. In
the other route, the same reactions were conducted in a dif-
ferent sequence. First, PPP with primary amine and PEG side
groups was formed by Suzuki reaction. The NCA ROP
through the pendant amino groups yielded the desired PPP
graft copolymers. The described approach via Suzuki cou-
pling and NCA ROP provides a versatile two-stage method
applicable for obtaining conjugated polymers and polypep-
tides. In principle, it should not matter which route is
employed first as both methods essentially yield structurally
identical polymers as evidenced by spectral characterization.
Highly emissive nature of the backbone and hydrophilic and
biocharacter of the side chains make these polymers an
interesting class of bio-related emitting materials to be fur-
ther evaluated in biosensor and functional materials. Such
applications were previously demonstrated for electrochemi-
cally formed polythiophene derivatives.30,39 Further studies
in this line are now in progress.
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