Side-chain glycylglycine-based polymer for simultaneous sensing and removal of copper(II) from aqueous medium

Article abstract of DOI:10.1002/pola.28968

Since glycylglycine (Gly-Gly) residue in the N-terminal region of human prion protein, a copper binding protein, binds with Cu(II), N-terminus Gly-Gly side-chain containing water soluble block copolymer was synthesized and used for simultaneous sensing an

Full text of DOI:10.1002/pola.28968

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Side-Chain Glycylglycine-Based Polymer for Simultaneous Sensing
and Removal of Copper(II) from Aqueous Medium
1
,2
1,2
2
1,2,3
Neha Choudhury, Sourav Mete, Srikanth Kambalapalli, Priyadarsi De
1
Department of Chemical Sciences, Polymer Research Centre, Indian Institute of Science Education and Research Kolkata,
Mohanpur, Nadia, West Bengal 741246, India
2
Department of Chemical Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, Nadia, West Bengal
7
41246, India
3
Department of Chemical Sciences, Centre for Advanced Functional Materials, Indian Institute of Science Education and Research
Kolkata, Mohanpur, Nadia, West Bengal 741246, India
Correspondence to: P. De (E-mail: p_de@iiserkol.ac.in)
Received 4 January 2018; accepted 17 January 2018; published online 00 Month 2018
DOI: 10.1002/pola.28968
ABSTRACT: Since glycylglycine (Gly-Gly) residue in the N-termi-
nal region of human prion protein, a copper binding protein,
binds with Cu(II), N-terminus Gly-Gly side-chain containing
water soluble block copolymer was synthesized and used for
simultaneous sensing and removal of Cu(II) ion from aqueous
medium. The polymer has amide nitrogen atom and ester car-
bonyl group as potential binding sites in the side-chain Gly-Gly
pendants. Job’s plot experiment confirms 2:1 binding stoichi-
ometry of polymer with Cu(II). This polymer is able to sense
parts per billion level of Cu(II) very selectively in an aqueous
medium and remove Cu(II) ions quantitatively by precipitating
out the Cu(II) via complex formation in the pH range 7–9. The
binding mode of polymer with Cu(II) in polymer-Cu(II) complex
1
was characterized by H NMR, FTIR, and UV–vis spectroscopy.
The attachment of Cu(II) in the polymer-Cu(II) complex was
confirmed by cyclic voltammetry experiment. Cu(II) release
from the complex was achieved at pH 5 due to the protonation
of amide nitrogen atoms in the Gly-Gly moiety. C
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0
KEYWORDS: block copolymers; Cu(II) ion; glycylglycine residue;
metal-polymer complexes; metal removal; sensors; water-solu-
ble block copolymer
1
3
INTRODUCTION Toxic heavy metal ions in water bring many
pernicious effects on environment and human health,
because of their toxicity and potential carcinogenicity. Sev-
eral techniques have been employed for the removal of toxic
heavy metal ions from aqueous medium such as complexa-
toxic heavy metal ions. Thus, polymeric materials contain-
ing amide, carboxylic acid, carbonyl, and amine groups can
1
2,3
1
4
be good polychelatogens and used for water purification.
Copper is an integral part of many essential enzymes and
glycoproteins, thus significantly important in biological sys-
tems. However, both copper excess and copper deficiency
will disrupt healthy metabolic function by creating mineral
In excess, and especially in nonorganically
bound form, copper has been linked to oxidative stress and
brain damage that may lead to cognitive decline and Alz-
4
5
tion and precipitation, reverse osmosis, electrochemical
1
5
6
7,8
9
treatment techniques, ion exchange, membrane filtration,
1
0
and adsorption. Among them, the complexation and precip-
itation technique is found to be a favorable method for
removal of toxic heavy metals with low concentration due to
1
6
imbalance.
1
1
its cost-effectiveness and high efficiency.
1
3
heimer’s disease.
Copper ions have been frequently
High solubility in aquatic environment and toxic nature of
heavy metal ions present in waste product of industries,
increase the demand of unique polymeric materials with the
purpose of separation and removal through complexation
and precipitation technique to restrict the environmental
pollution. Polychelatogens are metal ion chelating poly-
mers, contains one or more electron donor atoms such as N,
P, O, and S that can form coordinate bonds with most of the
detected in water streams and natural water, as Cu(II) is
widely used in wood pulp production, fertilizer industry,
paper and paper board mills, electroplating, electrical indus-
1
7,18
try, machinery, and many other industries.
The excess
amount of copper in water medium affect the self-
1
2
19
purification of bulk water and interrupt the microbiological
2
0
treatment of waste water. To protect consumers from the
effects of long term chronic exposure to copper compounds,
Additional Supporting Information may be found in the online version of this article.
2018 Wiley Periodicals, Inc.
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United States Environmental Protection Agency have pre-
scribed that the maximum contaminant level of Cu(II) in the
water used for consumption should not be more than 1.3
parts per million (1300 parts per billion). Therefore, the
detection of trace amount of Cu(II) and its removal from
aquatic environment, are very crucial for biological systems
and in terms of industrial point of view.
used to obtain molecular weights (MW) and MW distribu-
tions (dispersity, Ð) of polymers in tetrahydrofuran solvent
at 30 8C at 1.0 mL/min flow rate. The GPC instrument con-
tains a Waters 515 HPLC pump, a Waters 2414 refractive
index (RI) detector, one PolarGel-M guard column (50 3
7.5 mm) and two PolarGel-M analytical columns (300 3
7.5 mm). The FT-IR spectra were obtained on KBr disc with
a FTIR Perkin-Elmer RXI spectrometer at a nominal resolu-
2
1
The N-terminal region of human prion protein, a copper
binding protein, contains glycylglycine (Gly-Gly) residue for
the binding of Cu(II) through the deprotonated glycine amide
21
tion of 2 cm . The UV–vis spectroscopic measurements
were acquired on a Perkin–Elmer Lambda 35 spectropho-
tometer, with a scan rate of 120 nm/min. Cyclic voltammetry
was measured with a computer-controlled Princeton Applied
Research 263A electrochemical workstation using platinum
2
2
and glycine carbonyl. Hence, this Gly-Gly residue can be
used as a good precursor for the binding of Cu(II) ion.
Although, formation of Cu(II)-complex of Gly-Gly has been
(
Pt) disk as a working electrode, Pt wire as the counter elec-
2
3,24
studied in aqueous medium,
the binding was not selec-
trode and Ag/AgNO3 (10 mM in acetonitrile) as the refer-
ence electrode. The 0.1 M tetra-butylammoniumperchlorate
tive towards Cu(II). The chelate complexes of Gly-Gly with
Mn(II), Ni(II), Zn(II), Co(II), and Ca(II) have also been
(
TBAPC) was used as supporting electrolyte.
2
5,26
reported.
In the above metal-peptide chelate complexes
the potential binding sites were amine, amide carbonyl and
Monomer Synthesis
Synthesis of Boc-Gly-Gly-OH
2
0,23
carboxylate group.
Herein, we synthesized N-terminus
Gly-Gly based block copolymer that can selectively sense
Cu(II) ion in aqueous medium and remove it through com-
plexation and precipitation technique. This polymer forms a
metal-chelate complex with Cu(II) ion and precipitates out
from water. The presented block copolymer is cost-effective
and environment friendly, so it can be utilized for environ-
mental remediation and pollution control.
Boc protection of the amine group of Gly-Gly was performed
2
8
by following the literature procedure. Typically, a solution
of Gly-Gly (5.0 g, 38 mmol) in a mixture of dioxane (90 mL),
water (45 mL), and 1 M NaOH (45 mL) in a round bottom
flask was immersed in an ice-water bath and stirred. Then,
2
di-tert-butyl dicarbonate [(Boc) O] (9.1 g, 42 mmol) was
added to the solution drop-wise and stirring was continued
for 12 h at room temperature. The solution was concen-
trated in vacuum to about 20–30 mL, cooled in an ice-water
bath, covered with a layer of ethyl acetate (about 50 mL).
Then, the mixture was acidified with a dilute solution of
KHSO4 to pH 2–3. The aqueous phase was extracted with
ethyl acetate and this operation was performed several
times. The ethyl acetate extracts were separated, washed
with water and dried over anhydrous Na SO . Upon evapora-
EXPERIMENTAL
Materials
Gly-Gly (99%) and trifluoroacetic acid (TFA, 99.5%) were
purchased from Sisco Research Laboratories (SRL), India and
used as received. Dicyclohexylcabodiimide (DCC, 99%), 4-
dimethylaminopyridine (DMAP, 99%), 2,2’-azobis-(2-methyl-
2
4
0
propionitrile) (AIBN), anhydrous N,N -dimethylformamide
tion under vacuum, a white solid was obtained (4.25 g, 85%
(
DMF, 99.9%), 2-hydroxyethyl methacrylate (HEMA, 97%),
1
yield). H NMR (400 MHz, DMSO-d , d, ppm): 1.39 (C(CH ) ,
6
3 3
di-tert-butyl dicarbonate (99%), 1,4-dioxane (99%) were
purchased from Sigma-Aldrich and used without further
purification, except for AIBN, which was used after recrystal-
lization in methanol. The 4-cyano-(dodecylsulfanylthiocarbo-
nyl)sulfanylpentanoic acid (CDP) was synthesized using
9
1
3
H, s), 12.63–12.43 (COOH, 1H, br, s), 7.01–6.93 (NHCOO,
H, t), 8.07–8.00 (NHCO, 1H, t), 3.77–3.70 (CH COO, 2H, d),
2
.56–3.50 (CH NHCOO, 2H, d) (Supporting Information
2
Fig. S1).
2
7
literature procedure. The NMR solvents such as D O (99%
Synthesis of Boc-Gly-Gly-EMA
2
The synthesis of dipeptide based vinyl monomer, Boc-Gly-
Gly-EMA was performed by DCC/DMAP esterification of
HEMA and Boc-Gly-Gly-OH. Briefly, Boc-Gly-Gly-OH (6.0 g,
D), CDCl3 (99.8% D), methanol-d4 (99.8% D), and DMSO-d6
2
9
(
99% D) were purchased from Cambridge Isotope Laborato-
ries, USA. The solvents such as dichloromethane (DCM),
diethyl ether, hexanes (mixture of isomers), and ethyl acetate
were purified by following standard procedures. All the
experiments were performed using HPLC water purchased
from SRL, India.
2
1
5.84 mmol) was dissolved in 50 mL dry DCM, taken in a
00 mL round bottom flask and the solution was purged
with dry N . Then, a solution of DCC (5.86 g, 28.43 mmol)
2
and DMAP (0.63 g, 5.16 mmol) in 20 mL dry DCM was
added to the solution, immersed in an ice-water bath with
constant stirring. Next, HEMA (3.36 g, 25.84 mmol) was
added drop-wise to the reaction mixture. This reaction mix-
ture was allowed to react under the ice-water bath condition
for 30 min and then at room temperature for 24 h. After
Instrumentation
1
III
The H NMR spectra were recorded on a Bruker Avance
00 or a JEOL-FT NMR-AL spectrometer operating at 500
5
and 400 MHz, respectively. Positive mode electrospray ioni-
zation mass spectrometry (ESI-MS) was performed on a Q-
Tof Micro YA263 high resolution mass spectrometer (Waters
Corporation). Gel permeation chromatography (GPC) was
0
removing insoluble N,N -dicyclohexylurea by suction filtra-
tion, an additional 100 mL of distilled water was added to
the filtrate and then it was extracted 4 times with 120 mL of
2
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DCM. The organic layer was further washed with NaHCO3
and brine solution, and dried over anhydrous Na SO . The
and pH of the solution was adjusted to 7 with 4-(2-hydrox-
yethyl)-1-piperazineethanesulfonic acid) (HEPES buffer;
1 mM). The mixture was kept for stirring at room tempera-
ture for 24 h. After 24 h a green compound was precipitated
out from the solution. Then the precipitate was washed sev-
eral times with water to remove excess copper salt and poly-
mer. The solid compound was dried at 50 8C under high
vacuum for 24 h to a constant weight. Same procedure was
followed for the formation of complex at pH 9.
2
4
solvent was removed by rotary evaporation. It was further
purified by silica gel column chromatography using hexanes/
ethyl acetate as the mobile phase (4:1, v/v) to get a yellow
1
liquid Boc-Gly-Gly-EMA (obtained 4.2 g, 70% yield). H NMR
(
400 MHz, CDCl , d, ppm): 1.45–1.38 (C(CH ) , 9H, s), 1.93–
3 3 3
1.89 (C@CCH , 3H, s), 4.39–4.30 (OCH CH O, 4H, m), 6.11–
3 2 2
6
.07, and 5.59–5.55 (C@CH , 2H, s), 5.30–5.21 (NHCOO, 1H,
2
s), 6.75–6.68 (NHCO, 1H, s), 4.12–4.02 (CH COO, 2H, d),
2
3
.86–3.78 (CH NHCOO, 2H, s) (Supporting Information
RESULTS AND DISCUSSION
2
Fig. S2).
Monomer Synthesis
Synthesis of mPEG-Based Macro-CTA
The dipeptide based vinyl monomer, Boc-Gly-Gly-oxyethyl
methacrylate (Boc-Gly-Gly-EMA) was prepared by the esteri-
The mPEG2k/5k-CDP was synthesized following the previ-
3
0
ously established procedure.
fication reaction of Boc-protected dipeptide, Boc-Gly-Gly-OH
1
(
Supporting Information Scheme S1 and see H NMR spec-
Synthesis of Block Copolymer
trum in Supporting Information Fig. S1) with HEMA in the
presence of DCC and DMAP with 70% yield. The structure of
A typical polymerization procedure is as follows: Boc-Gly-
Gly-EMA (0.5 g, 1.45 mmol), mPEG5k-CDP (0.32 g, 5.81 3
1
Boc-Gly-Gly-EMA was confirmed by H NMR spectroscopic
22
23
10
mmol), AIBN (0.95 mg, 5.81 3 10
mmol; 0.1 mL
measurement (Supporting Information Fig. S2), where two
distinct peaks for vinyl protons at 6.11–6.07 and 5.59–
solution of 95 mg AIBN in 10 mL DMF) and DMF (1 mL)
were taken in a 20 mL septa sealed glass vial at a feed ratio
of [monomer]/[CTA]/[AIBN] 5 25:1:0.1. One small magnetic
stir bar was added to the vial, purged with dry N2 for 20
min, and the reaction vial was placed in a preheated block at
5.55 ppm, the methylene protons of oxyethyl methacrylate
group between 4.39 and 4.30 ppm and one methyl group
between 1.93 and 1.89 ppm range confirms the successful
attachment of HEMA with the Boc-protected dipeptide. Fur-
thermore, the structure of Boc-Gly-Gly-EMA was confirmed
70 8C for 9 h with constant stirring. The polymerization was
quenched by cooling the reaction vessel in an ice-water bath
followed by exposing the solution to air. The reaction mix-
ture was diluted with acetone and precipitated into cold hex-
anes. Then, the polymer was reprecipitated four times from
acetone/hexanes and dried under high vacuum at room tem-
perature for 6 h to a constant weight. The purified polymer
was isolated as yellow solid (obtained 425 mg, 85% yield).
by electrospray ionization mass spectrometry. (ESI-MS, m/z):
1
[
M 1 Na] calculated for Boc-Gly-Gly-EMA, 367.15; found,
367.15 (Supporting Information Fig. S3).
RAFT Polymerization Kinetics
To obtain homopolymers (P(Boc-Gly-Gly-EMA)) from Boc-
Gly-Gly-EMA with a desirable MW, reversible addition-
fragmentation chain transfer (RAFT) polymerization of Boc-
Gly-Gly-EMA was conducted in DMF at 70 8C using AIBN as
initiator and 4-cyano-(dodecylsulfanylthiocarbonyl)sulfanyl-
pentanoic acid (CDP) as the chain transfer agent (CTA;
Scheme 1). The polymerization reactions were carried out at
different ratios of monomer (M) to CDP, while the molar
ratios of [CDP]/[AIBN] 5 1:0.1 was kept constant. In all
homopolymerizations, unimodal RI traces shifted smoothly
towards lower elution volume with increasing conversion
with no evidence of high MW species from bimolecular ter-
mination [Fig. 1(A)]. As expected, increasing [M]/[CDP] ratio
gives polymers of higher MWs with narrow molar mass dis-
tributions (dispersity, Ð 5 1.13–1.28) [Fig. 1(B)]. The number
average MWs determined from gel permeation chromatogra-
phy (Mn,GPC) increased linearly with the [M]/[CDP]. However,
Mn,GPC values are somewhat lower compared with the theo-
retical number average MW (Mn,theo) determined from con-
version data using the following equation: Mn,theo 5 ([M]/
Synthesis of mPEG -b-P(NH -Gly-Gly-EMA)
5
k
2
Free primary amine groups were obtained by removal of
Boc-protecting groups from the side-chain of the mPEG -b-
5
k
P(Boc-Gly-Gly-EMA) block copolymer using TFA in DCM fol-
lowed by neutralization with NaHCO3 solution. In a typical
example, a 20 mL vial was charged with mPEG5K-b-(Boc-Gly-
Gly-EMA) (120 mg) and 1 mL DCM was added with constant
stirring for 10 min to maintain the homogeneity. Then, 1 mL
TFA was added drop-wise under ice-water bath condition
and was kept for stirring for 4 h at room temperature. Then,
the deprotected block copolymer was isolated by precipita-
tion from diethyl ether and dried under vacuum. After that,
the product was taken in a 20 mL vial and 1 mL H O was
2
added to it. The free amine containing block copolymer was
isolated by precipitation from saturated NaHCO3 solution
and dried under high vacuum at 50 8C for 24 h to a constant
weight. The polymer was isolated as yellow solid (obtained
84 mg, 70% yield).
[CDP] 3 MW of Boc-Gly-Gly-EMA 3 conversion) 1 (MW of
Synthesis of Polymer-Cu(II) Complex
CDP). This is possibly due to the different hydrodynamic vol-
ume of P(Boc-Gly-Gly-EMA) compared with the poly(methyl
methacrylate) standards, which we have used to construct
the conventional calibration curve for the determination of
Mn,GPC values from GPC study. The number average
Typically, in a 20 mL vial, mPEG -b-(NH -Gly-Gly-EMA)
5
k
2
2
3
(
50 mg, 7.29 3 10 mmol) was taken and 1 mL H O was
2
added to it. Then a solution of CuSO .5H O (1.82 mg, 7.29 3
4
2
23
10
mmol) in 1 mL H O was added to the polymer solution
2
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SCHEME 1 Synthetic scheme for the preparation of various polymers (2) from the Boc-Gly-Gly-EMA (1) via RAFT polymerization,
followed by deprotection of Boc-group and reaction of neutralized polymer (3) with Cu(II) salt in aqueous medium to form the cor-
responding complex (4). [Color figure can be viewed at wileyonlinelibrary.com]
molecular weight (Mn,NMR) of the homopolymers were also
calculated from H NMR spectroscopy by comparing the inte-
AOACH ACH AOA, ACH ACOOA, and ACH ANHACOOA
2 2 2 2
1
protons). The Mn,NMR values of 4500 and 7200 g/mol,
respectively, for the polymers obtained from [M]/
[CDP] 5 15:1 and 25:1 nicely matched with the correspond-
ing Mn,theo values, suggesting controlled synthesis of well-
defined homopolymers with high chain-ends fidelity.
gration areas from the terminal ACH ACH A protons (from
2
2
the HOOCACH ACH AC(CN)(CH )A chain end) at 2.4–
2
2
3
2.6 ppm and repeating unit protons at 3.78–4.39 ppm (for
mPEG-CDP Macro-CTA Derived Peptide Based Block
Copolymers
Side-chain Gly-Gly dipeptide-based block copolymers were
synthesized via RAFT polymerization technique, using mono-
methoxy poly(ethylene glycol) (mPEG2k and mPEG ) based
5k
macro-chain transfer agent [(mPEG)2k/5k-CDP] and AIBN as
initiator. The mPEG2k/5k-CDP was prepared following the
3
0
reported procedure. The formation of block copolymer was
1
confirmed by GPC and H NMR study. The attachment of
mPEG moiety to the block copolymer was confirmed by the
1
distinct peak at 3.79–3.59 ppm range in H NMR spectrum
(
Supporting Information Fig. S4). The Mn,NMR of the block
copolymer obtained from [M]/[mPEG -CDP] 5 25:1 ratio
5
k
was calculated as 7480 g/mol, by comparing the integration
areas from the ACH ACH A protons of the polymer chain
2
2
ends (for AOOCACH
2
ACH
2
AC(CN)(CH
3
) A protons) at 2.4–
2.6 ppm and repeating unit protons of side chain (for
AOACH ACH AOA, ACH ACOOA, and ACH ANHACOOA
2
2
2
2
protons) at 3.78–4.39 ppm (Supporting Information Fig. S4),
nicely matches with the Mn,GPC value of 7600 g/mol
(Ð 5 1.23; Supporting Information Fig. S5) but a little lower
than the theoretical MW (12,700 g/mol) determined based
on conversion. This Boc-protected block copolymer was
found to be highly soluble in water and most of the organic
solvents due to the presence of high MW mPEG moiety.
Deprotection of Boc-Group
Deprotection of Boc-group from Boc-protected block copoly-
3
0
mer was achieved using TFA in DCM at room temperature.
FIGURE 1 A: GPC RI traces of P(Boc-Gly-Gly-EMA) obtained at
different [M]/[CDP] ratios keeping constant [CDP]/[AIBN] 5 1:0.1
After that the protonated amine group of deprotected polymer
ratios and (B) dependence of M
the RAFT polymerization of Boc-Gly-Gly-EMA in DMF at 70 8C.
Color figure can be viewed at wileyonlinelibrary.com]
n
and Ð on [M]/[CDP] ratios, for
was neutralized using NaHCO solution. Successful deprotona-
tion and neutralization was confirmed by disappearance of
Boc-proton signal in the range 1.45–1.38 ppm and appearance
3
[
4
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1
FIGURE 2 H NMR spectra of (A) mPEG5k-b-P(NH
2
-Gly-Gly-
24
EMA) in D
addition of Cu(II) salt (10 M) in D
complex of polymer in 1:1 mixture of D
2
O, (B) neutralized polymer (10
M) after instant
O at pD 5 7, and (C) Cu(II)-
O and DMSO-d
Color figure can be viewed at wileyonlinelibrary.com]
24
2
2
6
.
[
2
of ANH proton peak at 8.65 ppm, respectively (Supporting
Information Fig. S6). The neutralized block copolymer was sol-
uble in water. The mPEG2k-b-P(NH -Gly-Gly-EMA) block copol-
ymer was also prepared. Due to the lower aqueous solubility
of mPEG2k-b-P(NH -Gly-Gly-EMA), we used mPEG5k-b-P(NH
2
2
2
-
Gly-Gly-EMA) for all further experiments.
Characterization of Polymer-Cu(II) Complex
Herein, we used the free amine containing block copolymer for
simultaneous sensing and removal of Cu(II) ion from aqueous
solution. The block copolymer, mPEG -b-P(NH -Gly-Gly-EMA)
FIGURE 3 A: FTIR spectra of mPEG5k-b-P(NH
2
-Gly-Gly-EMA)
and polymer-Cu(II) complex and (B) UV–vis spectra of polymer
and polymer-Cu(II) complex. [Color figure can be viewed at
wileyonlinelibrary.com]
5
k
2
forms a polymeric metal-chelate complex with Cu(II) ion
Scheme 1) at pH 7–9 (Supporting Information Fig. S7). To
(
1
ensure the attachment of Cu(II) in the polymeric side chain, H
NMR spectrum of the polymer-Cu(II) complex was recorded and
compared with the corresponding spectrum of N-terminus block
present in the block copolymer (Supporting Information
Fig. S6), confirms no binding interaction between between
Cu(II) and ANH group of the polymer in the polymer-Cu(II)
2
2
4
copolymer (Fig. 2). After the instant addition of Cu(II) (10 M)
complex. Thus, complex formation happens by attachment of
Cu(II) with the amide nitrogen atom and oxygen atom of the
ester carbonyl group of the polymeric side chain. The poly-
mer-Cu(II) complex was insoluble in most of the organic sol-
vents except in dimethyl sulfoxide and acetonitrile, where
the complex was sparingly soluble. The insolubility of the
resultant polymer-Cu(II) complex was attributed to the inter-
molecular cross-linking among the side-chain Gly-Gly pend-
ants in the polymeric building block.
24
in D O to the polymer (10 M) solution in D O at pD5 7, a
2
2
shielding of methylene proton (OCACH ANHA) attached in
2
between the amide nitrogen atom and ester carbonyl group takes
place due to the attachment of Cu(II) in the side chain, which
cause a shifting of the peak from 4.48–4.38 ppm region
[
Fig. 2(A)] to 4.30–4.15 ppm region [Fig. 2(B)]. As shown in the
1
H NMR spectrum of polymer-Cu(II) complex in 1:1 mixture of
D O and DMSO-d , these methylene protons shielded further and
2
6
merge with adjacent methylene protons (OACH ACH AO) pre-
2
2
The comparison of significant FT-IR bands of neutralized
sent in the side chain of the polymer [Fig. 2(C)]. The amide pro-
ton peak of the block copolymer between 8.2 and 8.0 ppm
completely disappeared in the complex, indicating deprotonation
of the amide nitrogen atom during complex formation.
block copolymer, mPEG -b-P(NH -Gly-Gly-EMA) and poly-
5
k
2
mer-Cu(II) complex used further to determine the ligand
mode of coordination with Cu(II) ion [Fig. 3(A)]. Two bands
2
1
at 3434 and 3465 cm for NAH stretching vibration of pri-
mary amine group of the polymer retained at the same posi-
tion in polymer-Cu(II) complex, indicating no interaction
The retention of ANH proton peak at 8.65 ppm in the poly-
2
meric complex [Fig. 2(C)], without any shifting, as it was
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FIGURE 4 Cyclic voltammogram of polymer, Cu(II) salt and
polymer-Cu(II) complex in 1:1 mixture of acetonitrile and H
containing 0.1 M TBAPC, using Pt disk as a working electrode,
Pt wire as the counter electrode and Ag/AgNO (10 mM in ace-
FIGURE 5 Bar diagram representation of selective response of
2
O
the polymer for Cu(II) by UV–vis spectroscopy.
3
Electrochemical behavior of mPEG -b-P(NH -Gly-Gly-EMA)
5
k
2
tonitrile) as the reference electrode, scan rate 100 mV/s. [Color
figure can be viewed at wileyonlinelibrary.com]
and corresponding polymer-Cu(II) complex was studied by
cyclic voltammetry (CV) at a Pt electrode, using 1:1 acetoni-
trile and H O mixture (volume/volume). In Figure 4, CV run
did not show any oxidation and reduction peaks for the neu-
tralized mPEG5k-b-P(NH -Gly-Gly-EMA) polymer, revealing its
2
2
between primary amine group and Cu(II) in the polymeric
21
side chain. The band at 1708 cm for C@O stretching vibra-
21
tion of ester linkage of the polymer shifted to 1768 cm
electro-inactivity in aqueous solution at pH 7. The Cu(II) salt
gave peaks at 10.22 V for Cu(II)/Cu(I) reduction process
and peak at 10.49 V for Cu(I)/Cu(II) oxidation process in
the cyclic voltammogram whereas, the polymer-Cu(II) com-
plex showed peaks at 10.42 V for Cu(II)/Cu(I) reduction
process and 10.15 V for Cu(I)/Cu(II) oxidation process
after complex formation and similarly CAO stretching vibra-
tion of ester linkage also shifted from 1091 to 1154 cm
21
indicating strong interaction between ester carbonyl group
with Cu(II) in the polymer-Cu(II) complex. The small shifting
2
1
of FT-IR band from 1618 cm for C@O stretching vibration
2
1
of amide linkage to 1649 cm after complex formation was
due to the attachment of adjacent amide nitrogen atom with
Cu(II) ion in the polymer-Cu(II) complex.
(
Fig. 4). Lower value of reduction potential for the polymer-
Cu(II) complex than that for free Cu(II) ion, suggesting that
reduction of Cu(II) to Cu(I) becomes difficult in the complex.
It was interesting to observe from the cyclic voltammogram,
that the polymer-Cu(II) complex showed metal centered
Cu(II)/Cu(I) reversible redox couple. This experiment further
confirms the attachment of Cu(II) in the polymer to form the
polymer-Cu(II) complex.
The UV–vis spectra of both block copolymer and metal complex
shows an absorption maxima, kmax 5 303 nm for trithiocarbon-
ate moiety, indicating the retention of x-trithiocarbonate
3
0
group, during RAFT polymerization and after complex forma-
tion. One new peak having absorption maxima at 635 nm was
observed in the UV–vis spectra of polymer-Cu(II) complex
which confirms the attachment of Cu(II) with the block copoly-
Selectivity of the Block Copolymer for Cu(II)
3
1
In the next stage, selectivity of the polymer for Cu(II) detec-
tion was investigated. A variety of competitive metal ions,
including Fe(III), Co(II), Ni(II), Mn(II), Hg(II), As(III), Zn(II),
mer [Fig. 3(B)]. At pH 6, no complex formation takes place
and the extent of complex formation increases with the
increase of pH from 7 to 9 (Supporting Information Fig. S7)
due to the increased rate of deprotonation of amide nitrogen
atom in the side-chain. At pH 5, this polymer complex releases
the Cu(II) ion and solubilize in water due to the protonation of
amide nitrogen atoms confirmed by the disappearance of
absorbance at 635 nm in UV–vis spectra (Supporting Informa-
tion Fig. S8). For further verification of the binding site, we
added Cu(II) solution to the aqueous solution of Boc-protected
block copolymer mPEG -b-P(Boc-Gly-Gly-EMA) and UV–vis
23
Cu(II), and Cr(III) (with concentration 10
M in water),
were individually added to the polymer solution (with con-
2
3
centration 10 M in water) at pH 7 and absorption values
were recorded by UV–vis spectroscopy (Supporting Informa-
tion Fig. S9). The addition of other metals to the solutions of
mPEG5k-b-P(NH -Gly-Gly-EMA) resulted in little or no effect
2
on polymer. The absorbance at 635 nm for different metal
ions represented by a bar diagram (Fig. 5), which further
confirms the excellent selectivity of the polymer for Cu(II)
detection. Note that it is difficult to precisely predict the rel-
ative affinity of a specified ligand for a particular metal ion
due to combination of ligand field stabilization, the ability of
5
k
spectrum was recorded. We observed the same absorption
maxima at 635 nm in the UV–vis study, which once again con-
firms no interaction between the terminal nitrogen atom of the
side chain and Cu(II) during the complex formation.
6
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FIGURE 6 Job’s plot of polymer (based on repeating unit) with
FIGURE 8 Study for the removal efficiency of the polymer for
Cu(II) by UV–vis spectroscopy. [Color figure can be viewed at
wileyonlinelibrary.com]
Cu(II) in aqueous medium.
the metal ion to deprotonate the amide nitrogen, changes in
complex geometry during coordination, steric effects in the
peptide, and presence of soft coordination points on the
against Cu(II) concentration (Fig. 7). A detection limit of 317
parts per billion (5 lM) of Cu(II) was observed using our
unique polymer. These results indicate that this polymer can
be potentially used to detect Cu(II) in aqueous solution with
high sensitivity.
3
2
amino acid side chains.
Derivation of the Binding Stoichiometry
To confirm the binding stoichiometry between the Cu(II) and
peptide side-chain, the Job’s plot experiment was carried
out at a total concentration of 10 M of the metal ion and
the polymer (based on repeating unit) at pH 7 using HEPES
buffer (1.0 mM). This continuous variation method indicated
a 2:1 binding of the polymer with Cu(II) ion (Fig. 6).
3
3
2
4
Removal Efficiency of the Block Copolymer towards
Cu(II)
The removal efficiency of the polymer was further investi-
gated by UV–vis spectroscopy. Several samples having same
concentration of Cu(II) (0.015 mmol) were prepared. Then,
they were reacted with polymer solutions having different
concentration (based on repeating unit) from 0.1 to 3.5
equivalent (taking metal concentration as 1 equivalent) at
pH 7 and absorbance value at 635 nm was recorded (Fig. 8).
The plot of absorbance versus ratio of equivalent concentra-
tion of monomer repeating unit and Cu(II), indicates a satu-
ration after 2.3 equivalent concentration of polymer. This
saturation indicates that for the complete removal of 1
equivalent metal (1.0 mg, 0.015 mmol), 2.3 equivalent poly-
mer (55 mg, 0.034 mmol) was needed. This result confirms
the high removal efficiency of the polymer towards
Cu(II) ion.
Sensitivity of the Block Copolymer for Cu(II)
For the sensitivity study, polymer solutions were treated
with varying concentration of Cu(II) from 1 to 650 lM at pH
7
and absorbance values at 635 nm were recorded (Fig. 7).
Absorbance value at 635 nm increased significantly with the
increase of Cu(II) concentration. A straight line was found in
the calibration curve by plotting absorbance at 635 nm
CONCLUSIONS
In conclusion, we have successfully developed a Gly-Gly
dipeptide-based water soluble block copolymer having con-
trolled MW and narrow Ð with high sensitivity, selectivity
and removal efficiency for Cu(II) in aqueous solution. The
amide nitrogen atom and oxygen atom of ester carbonyl
group from two Gly-Gly side-chains binds with a Cu(II) to
form a 2:1 polymeric metal chelate complex at pH 7–9.
Release of Cu(II) from the complex was achieved at pH 5
due to the protonation of amide nitrogen atoms in the Gly-
Gly moiety, which suggests reversible binding of Cu(II). The
complex formation takes place at physiological pH, so it can
be used in environmental and other applications.
FIGURE 7 Calibration curve based on UV–vis study of block
copolymer with known Cu(II) concentration. [Color figure can
be viewed at wileyonlinelibrary.com]
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ACKNOWLEDGMENTS
15 A. E. G. Cass, A. E. O. Hill, In Ciba Foundation Symposium
9-Biological Roles of Copper; D. Evered, G. Lawrenson, Eds.;
Wiley: Chichester, UK, 1980; Chapter 5, pp 71.
7
N. Choudhury and S. Mete acknowledge Indian Institute of Sci-
ence Education and Research Kolkata, and Council of Scientific
and Industrial Research (CSIR), Government of India, respec-
tively, for their research fellowship. The authors acknowledge
Kamal Bauri for fruitful discussions.
1
1
1
6 K. C. Chow, L. M. Gaetke, Toxicology. 2003, 189, 147.
7 V. K. Gupta, Ind. Eng. Chem. Res. 1998, 37, 192.
8 A. Troupis, A. Hiskia, E. Papaconstantinou, Environ. Sci.
Technol. 2002, 36, 5355.
1
1
9 T. Yubin, C. Fangyan, Z. Honglin, Adsorp. Sci. Tech. 1998,
6, 595.
REFERENCES AND NOTES
2
0 S. E. Ghazy, S. E. Samra, S. M. El-Morsy, Adsorp. Sci. Tech.
1
2
J. O. Nriagu, J. M. Pacyna, Nature. 1988, 333, 134.
2001, 19, 175.
M. A. Peraza, F. Ayala-Fierro, D. S. Barber, E. Casarez, L. T.
21 R. Dixit, Wasiullah, D. Malaviya, K. Pandiyan, U. Singh, A.
Sahu, R. Shukla, B. Singh, J. Rai, P. Sharma, H. Lade, D. Paul,
Sustainability. 2015, 7, 2189.
Rael, Environ. Health Perspect. 1998, 106, 203.
3
G. S. Shukla, R. L. Singhal, Can. J. Physiol. Pharmacol. 1984,
6
2, 1015.
22 C. S. Burns, E. Aronoff-Spencer, C. M. Dunham, P. Lario, N.
I. Avdievich, W. E. Antholine, M. M. Olmstead, A. Vrielink, G. J.
Gerfen, J. Peisach, W. G. Scott, G. L. Millhauser, Biochemistry.
4
H. Sakai, S. Matsuoka, A. A. Zinchenko, S. Murata, Colloids
Surf. A 2009, 347, 210.
2
2
2
2
002, 41, 3991.
5
H. Ozaki, K. Sharm, W. Saktaywin, Desalination. 2002, 144,
3 M. Sheinblatt, E. D. Becker, J. Biol. Chem. 1967, 242, 3159.
4 R. Nakon, R. J. Angelici, Inorg. Chem. 1973, 12, 1269.
5 J. B. Hodgson, G. C. Percy, J. Mol. Struct. 1977, 37, 193.
287.
6
7
L. J. J. Janssen, L. Koene, Chem. Eng. J. 2002, 85, 137.
M. Y. Vilensky, B. Berkowitz, A. Warshawsky, Environ. Sci.
Technol. 2002, 36, 1851.
26 M. J. A. Rainer, B. M. Rode, Monatsh. Chem. 1982, 113, 391.
8
2
Y. Q. Xing, X. M. Chen, D. H. Wang, Environ. Sci. Technol.
007, 41, 1439.
27 G. Moad, Y. K. Chong, A. Postma, E. Rizzardo, S. H. Thang,
Polymer. 2005, 46, 8458.
9
R. S. Juang, R. C. Shiau, J. Membr. Sci. 2000, 165, 159.
28 P. Jana, S. Maity, S. K. Maity, D. Haldar, Chem. Commun.
2
011, 47, 2092.
10 K. Kadirvelu, C. Faur-Brasquet, P. L. Cloirec, Langmuir. 2000,
16, 404.
29 S. Kumar, S. G. Roy, P. De, Polym. Chem. 2012, 3, 1239.
1
1 X. Jiang, C. Peng, D. Fu, Z. Chen, L. Shen, Q. Li, T. Ouyang,
30 S. Kumar, R. Acharya, U. Chatterji, P. De, J. Mater. Chem. B.
2013, 1, 946.
Y. Wang, Appl. Surf. Sci. 2015, 353, 1087.
1
2 S. Stevanovic, K. Trivunac, Chemosphere. 2006, 64, 486.
3 A. E.-H. Ali, H. A. Shawky, H. A. Abd El Rehim, E. A. Hegazy,
31 S. C. Lee, M. R. Iztt, X. X. Zhang, G. E. Nelson, D. T. Lamb,
B. P. Savage, S. J. Bradshow, Inorg. Chim. Acta. 2001, 317,
1
1
74.
Eur. Polym. J. 2003, 39, 2337.
4 E. K. Yetimoglu, M. V. Kahraman, O. Ercan, Z. S. Akdemir,
3
2 J. J. Gooding, D. B. Hibbert, W. Yang, Sensors. 2001, 1, 75.
1
N. K. Apohan, React. Funct. Polym. 2007, 67, 451.
33 S. Bhattacharya, R. Sunmugam, RSC Adv. 2015, 5, 74973.
8
JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY 2018, 00, 000–000