l-Histidine-Derived Smart Antifouling Biohybrid with Multistimuli Responsivity

Article abstract of DOI:10.1021/acs.biomac.1c00748

A novel dual pH/thermoresponsive amphiphilic poly(histidine methacrylamide)-block-hydroxyl-terminated polybutadiene-block-poly(histidine methacrylamide) (PHisMAM-b-PB-b-PHisMAM) triblock copolymer biohybrid, composed of hydrophobic PB and ampholytic PHisMAM segments, is developed via direct switching from living anionic polymerization to recyclable nanoparticle catalyst-mediated reversible-deactivation radical polymerization (RDRP). The transformation involved in situ postpolymerization modification of living polybutadiene-based carbanionic species, end-capped with ethylene oxide, into dihydroxyl-terminated polybutadiene and a subsequent reaction with 2-bromo-2-methylpropionyl bromide resulting in a telechelic ATRP macroinitiator (Br-PB-Br). Br-PB-Br was used to mediate RDRP of an l-histidine-derived monomer, HisMAM, yielding a series of PHisMAM-b-PB-b-PHisMAM triblock copolymers. The copolymer’s stimuli response was assessed against pH and temperature changes. The copolymer is capable of switching among its zwitterionic, anionic, and cationic forms and exhibited unique antifouling properties in its zwitterionic form. These novel triblock copolymers are expected to be show promising potential in biomedical applications.

Full text of DOI:10.1021/acs.biomac.1c00748

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Article
L‑Histidine-Derived Smart Antifouling Biohybrid with Multistimuli
Responsivity
Sk Arif Mohammad, Subrata Dolui, Devendra Kumar, Shivshankar R. Mane, and Sanjib Banerjee*
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ABSTRACT: A novel dual pH/thermoresponsive amphiphilic
poly(histidine methacrylamide)-block-hydroxyl-terminated polybu-
tadiene-block-poly(histidine methacrylamide) (PHisMAM-b-PB-b-
PHisMAM) triblock copolymer biohybrid, composed of hydro-
phobic PB and ampholytic PHisMAM segments, is developed via
direct switching from living anionic polymerization to recyclable
nanoparticle catalyst-mediated reversible-deactivation radical poly-
merization (RDRP). The transformation involved in situ
postpolymerization modification of living polybutadiene-based
carbanionic species, end-capped with ethylene oxide, into
dihydroxyl-terminated polybutadiene and a subsequent reaction
with 2-bromo-2-methylpropionyl bromide resulting in a telechelic
ATRP macroinitiator (Br−PB−Br). Br−PB−Br was used to mediate RDRP of an L-histidine-derived monomer, HisMAM, yielding a
series of PHisMAM-b-PB-b-PHisMAM triblock copolymers. The copolymer’s stimuli response was assessed against pH and
temperature changes. The copolymer is capable of switching among its zwitterionic, anionic, and cationic forms and exhibited unique
antifouling properties in its zwitterionic form. These novel triblock copolymers are expected to be show promising potential in
biomedical applications.
low viscosity, good transparency, oil resistance, and ease of
processability.8 Thus, a combination of the properties of
polybutadiene with those of an amino acid-based zwitterionic
polymer may lead to a biohybrid with unique properties
derived from the synergy of properties of these two individual
blocks.
Recent developments in the reversible-deactivation radical
polymerization (RDRP) technique have revolutionized the
field of precision polymer synthesis, providing access to well-
defined functional (co)polymers with high end group fidelity
and complex macromolecular architectures.9 Some of the
widely used RDRP techniques are atom transfer radical
polymerization (ATRP),10−12 single-electron transfer living
radical polymerization (SET-LRP),13,14 reversible addition−
fragmentation chain transfer (RAFT) polymerization,15,16
organometallic-mediated radical polymerization,17,18 organo-
heteroatom-mediated radical polymerization,19 and nitroxide-
mediated radical polymerization (NMP).20 Some interesting
block copolymers with unique properties have been synthe-
1. INTRODUCTION
Nature has developed several strategies utilizing the receptive
properties of highly functional protein molecules to efficiently
perform a variety of complex biological functions. Notably,
many biological processes, including but not limited to
controlled complex enzyme-mediated reactions, can be
induced by temperature1 or pH.2 The development of novel
synthetic analogues that mimic the nature’s designs and are
capable of responding to physical and chemical changes in
their environment, such as temperature and light,3 may enable
new advanced applications. Covalent attachment of synthetic
polymers with various biomolecules (such as poly(amino
acid)/peptide/protein) affords facile access to biohybrids,
which combine benefits from both the synthetic polymer world
and nature.4 These hybrids possess some unique properties
such as excellent biodegradability and biocompatibility as well
as the ability to self-assemble into well-defined three-
dimensional nanostructures. Recently, these biohybrids have
been attracting considerable attention for the development of
environment friendly smart materials for application in
engineering and biomedical sciences.5 Among these, amino
acid-based zwitterionic polymers6,7 are very attractive to
researchers as they show some unique properties such as
water solubility, low toxicity, biocompatibility, and biodegrad-
ability. On the other hand, hydroxyl-terminated polybutadiene
(HTPB), a low molecular weight telechelic rubber, has been
used in many engineering and material applications due to its
Received: June 11, 2021
Revised: July 17, 2021
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Scheme 1. Synthesis of PHisMAM-b-PB-b-PHisMAM Triblock Copolymer Biohybrids via Mechanistic Transformation of
Living Anionic Polymerization into RDRP
sized using controlled polymerization techniques.21,22 Materi-
als with antifouling properties23−28 are indispensable for a wide
range of biomedical applications. Polyethylene glycol (PEG)
has been the most widely used antifouling agent in
biotechnology, surface science, packaging, and chemical
engineering.29 However, PEG is nonbiodegradable and leads
to bioaccumulation.30 Thus, there is a need to develop
alternative antifouling materials for biotechnological and
biomedical applications. Zwitterionic polymers have excellent
antifouling ability and have been postulated as a new
generation of antifouling materials.31 To this end, researchers
have developed natural amino acid (L-cysteine32,33 and L-
serine34)-based zwitterionic polymers that are sometimes
employed to develop antifouling materials.
(histidine methacrylamide) (PHisMAM-b-PB-b-PHisMAM)
triblock copolymer biohybrid, composed of L-histidine
(ampholyte) and butadiene (hydrophobic moiety),39 via
recyclable (up to multiple cycles) alloy-mediated ambient
temperature RDRP of an L-histidine-derived monomer,
HisMAM, using a telechelic bromo-terminated polybutadiene
(Br−PB−Br) macroinitiator (Scheme 1). Br−PB−Br was
synthesized via living anionic polymerization of butadiene
followed by postpolymerization modification. These novel
PHisMAM-b-PB-b-PHisMAM triblock copolymers are ex-
pected to show promising potential for biomedical applications
and demonstrate an antifouling behavior.
2. EXPERIMENTAL SECTION
However, some of these materials contain toxic thiol32 or
synthesized via RAFT34 polymerization. Also, there is a
plethora of reports on the synthesis of amino acid-based
polymers (which does not exhibit antifouling properties) via
different RDRP techniques such as ATRP, NMP, and RAFT
polymerization.35 However, the synthesis of such polymers
suffer from major drawbacks: (a) difficulty in recovery of the
catalyst residues from the final products, which leads to their
coloration and sometimes toxicity (for ATRP),36 (b)
undesired color, odor, and toxicity due to S-chain ends (for
RAFT polymerization),37 and (c) difficulty in the separation of
residual Cu(I) and/or Cu(II) precursors (used for in situ
generation of Cu(0) and Cu(0) from the final polymers (for
SET-LRP).38 Considering the ever expanding interest in amino
acid-based stimuli-responsive antifouling polymers toward
applications in biological and therapeutic fields, there is a
need to develop a green synthesis protocol for the construction
of a novel amino acid-based multifunctional network with
antifouling properties and tunable stimuli-responsive proper-
ties for applications in emerging areas.
2.1. Experimental Procedures. 2.1.1. Materials. Cobalt(II)
acetylacetonate (Co(acac)2, 97%, Aldrich), nickel(II) acetylacetonate
(Ni(acac)2, 95%, Aldrich), hydrazine hydrate (N2H4 H2O, reagent
grade, N2H4 50−60%, Aldrich), poly(vinylphenol) (PVPh, Mw
∼
11,000, Aldrich), ethyl α-bromoisobutyrate (EBiB, 98%, Aldrich), 2-
bromo-2-methylpropionyl bromide (BiBB, 98%, Aldrich), triethyl-
amine (TEA, ≥99%, Aldrich), tris[2-(dimethylamino)ethyl]-amine
(Me6TREN, >98%, TCI Chemicals), L-histidine (His, ≥99%,
Aldrich), N-isopropylacrylamide (NIPAM, 99%, TCI Chemicals),
poly(ethylene glycol) (PEG, Mn 400, Aldrich), bovine serum albumin
(BSA, ≥98.0%, Aldrich), methacryloyl chloride (Avra, India, purity
95%), 8-hydroxyquinoline (TCI Chemicals, purity >99%), sodium
hydroxide (NaOH, 96%, Finar, India), sodium nitrate (NaNO2, 97%,
Finar, India), potassium carbonate (K2CO3, ≥98.0%, Aldrich),
sodium chloride (NaCl, 98%, Finar, India), and hydrochloric acid
(HCl, 12 N, Finar, India) were used as received. Milli-Q water of
specific resistivity of 18.2 ΜΩ cm at 25 °C was used in all the
experiments. Methanol (MeOH, Merck, India), ethyl acetate (Merck,
India), and ethanol (Merck, India) were purified by distillation just
before use in the polymerization. Deuterium oxide (D2O) used for
nuclear magnetic resonance (NMR) spectroscopy was purchased
from Aldrich (purity >99.9%). Cyclohexane was purchased from
Sigma-Aldrich and purified by stirring over sulfuric acid and distilled
over styryl lithium. MeOH and tetrahydrofuran (THF) were
purchased from Spectrochem and used without further purification.
Herein, we report for the first time the synthesis of a novel
ABA-type amphiphilic dual pH/thermoresponsive poly-
(histidine methacrylamide)-block-polybutadiene-block-poly-
B
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n-Butyl lithium, 3-chloropropanol, styrene, t-butyldimethylsilyl
chloride, and imidazole were purchased from Sigma-Aldrich and
used as received. Butadiene was purchased from Sigma-Aldrich and
purified by distillation over n-butyl lithium. Ethylene oxide was
purchased from a local vendor and purified by distillation over n-butyl
lithium.
7.2 mmol) in dichloromethane was added for over 10 min. The
reaction mixture was stirred for 12 h at room temperature. The
reaction mixture was diluted with 50 mL of dichloromethane and
washed with a saturated solution of NH4Cl. The aqueous phase was
extracted with dichloromethane. The combined organic phase was
washed with brine and dried over Na2SO4. The crude product was
purified by fractional distillation to obtain 3-chloro-1-t-butyldime-
thylsilyloxy propane as a colorless liquid (yield 85%).
2.1.2. Purification of Reagents. 2.1.2.1. Butadiene. This monomer
is a gas at room temperature, so it is condensed into a flask (A) that
contains n-butyl lithium (n-BuLi) at −78 °C (dry ice/isopropanol
bath). The monomer is stirred for 20−30 min at 10 °C (ice/salt
bath). Then, it is distilled into another flask (B) containing fresh n-
BuLi and allowed to stand for 20−30 min at 10 °C until the viscosity
is slightly increased indicating the absence of active impurities.
2.1.2.2. Ethylene Oxide. Ethylene oxide is a low boiling compound,
and it is initially condensed into a cylinder containing freshly ground
calcium hydride (CaH2). It was stirred over CaH2 on a vacuum line
for about 30 min at 0 °C and distilled under a reduced pressure.
Ethylene oxide was further distilled over n-BuLi using the freeze−
pump−thaw technique. During its exposure to n-BuLi, ethylene oxide
was kept at 0 °C with stirring for 30 min. Finally, the pure monomer
was distilled into a graduated cylinder and stored at 20 °C until
further use.
2.1.2.3. Cyclohexane. Purification of cyclohexane was carried out
by treating with sulfuric acid for 1 week and then stirring over CaH2.
It was then degassed and distilled under vacuum in a reservoir
containing n-BuLi with styrene as an indicator. Presence of the orange
color indicates that the solvent is moisture-free and can be used for
polymerization.
2.1.3. Synthesis of Ni−Co Alloy Nanoparticles. Ni−Co alloy
nanoparticles were prepared by following a procedure reported
earlier.40 Typically, Co(acac)2 (0.005 mol) and Ni(acac)2 (0.005
mol) were added to a solution of PVPh in DMSO (0.2 wt %) under a
N2 atmosphere and then N2H4 H2O solution and NaOH solution
(0.02 M) were added to the solution rapidly and the mixture was
stirred magnetically at 70 °C for 2 h. After the reaction, the solution
was allowed to cool down to room temperature and a mixture of
water and ethanol (1:2 v/v) was added to it. The prepared alloy
nanoparticles were isolated using a bar magnet and washed thrice with
a water/ethanol mixture to remove unreacted starting materials. This
purification process was repeated several times to remove the excess
reactants and finally we performed drying under vacuum to obtain the
Ni−Co alloy nanoparticle.
1H NMR (CDCl3) δ (ppm) (Figure S3): 0.1−0.2 (−CH3 of silyl
6H), 0.82 (−CH3 of silyl 9H), 3.8 (−OCH2, 2H), 3.6 (−CH2−Cl,
2H), and 1.9 (−CH2−, 2H).
Step II t-Butyldimethylsilyloxy-1-propyllithium
In a 250 mL three-neck round-bottom flask equipped with a
Teflon-coated stir bar, dry cyclohexane (50 mL) and freshly cut pieces
of lithium (0.804 g, 0.114 mol) were added under an inert
atmosphere. Then, t-butyldimethylsilyloxy-1-chloropropane (2.0 g,
9.1 mmol) was added dropwise at 40 °C over 30 min. Upon complete
addition, the reaction mixture was heated to 60 °C and stirred for 16
h. The reaction mixture was cooled to room temperature and
transferred to a Schlenk flask (yield 70%).
2.1.5.2. Synthesis of a Monohydroxyl-Terminated Polybutadiene
Polymer. To a polymerization assembly, cyclohexane (200 mL) was
transferred followed by addition of butadiene (10 g) under an ice-cold
condition under constant stirring. An appropriate amount of t-
butyldimethylsilyloxy-1-propyllithium was then transferred (1.5 g) to
the above solution. The reaction mixture was then stirred at room
temperature for 10 min and then at 60 °C for 2 h. A small aliquot was
removed for characterization. Excess of ethylene oxide was then added
by transfer and polymerization was continued for the next 2 h. An
appropriate amount of MeOH was then added to the reaction mass,
and it was cooled to room temperature. The HTPB polymer was then
isolated by precipitation in MeOH.
1H NMR (CDCl3) δ (ppm) (Figure S4): 0.1−0.2 (−CH3 of silyl
6H), 0.82 (−CH3 of silyl 9H), 1.2−2.4 (−CH2 of a linear chain
proton), 3.4−3.6 (−OCH2), 4.9−5.1 (−CHCH2 of a vinyl
proton), and 5.25−5.75 (−CHCH− of a linear chain proton).
Degree of polymerization (DP)calculation
For the monohydroxyl-terminated polybutadiene polymer, which
was subsequently converted to Br−PB−Br and used as a macro-
initiator for HisMAM polymerization, the DP value was calculated
1
using H NMR (Figure S4) using the following equations
(A) Proton No. 1, 2’s total 2 protons and its integration value is
108.37.
2.1.4. Synthesis of L-Histidine Methacrylamide (HisMAM).
HisMAM was prepared following a modification of a procedure
reported earlier.41 Typically, L-histidine (500 mg) and NaNO2 (20
mg) were dissolved in K2CO3 aqueous solution (3.0 mL, 5%, v/v) and
the solution was cooled down to 0 °C in an ice bath. Methacryloyl
chloride (400 μL) was added to the aqueous solution of L-histidine
dropwise under a nitrogen atmosphere at 0−5 °C. After mixing, the
reaction mixture was stirred continuously for 2 h at room
temperature. After the reaction, the unreacted chemicals and
byproducts were removed by extraction with ethyl acetate. The pH
of the aqueous solution was adjusted to 5 by 2 N NaOH, and the
product was extracted with ethanol to remove excess L-histidine and
NaCl. The ethanol layer was then evaporated, and the product was
dissolved in ethanol and precipitated in acetone. The precipitate was
isolated and vacuum-dried for 12 h to obtained HisMAM.
1H NMR (D2O) δ (ppm) (Figure S2): 1.85 (CH3C(CH2)),
2.85−3.30 (−CH2−imidazole), 4.40−4.50 (−NHCH(COOH)-
CH2−), 5.40−5.60 (−CH2C(CH3)−), 7.10 (1H imidazole, −C
CHN)), and 8.10 (1H imidazole, −NCHNH−).
A = [108.37/2] = 54.18
(1)
(B) Proton No. 3, 4’s total 3 protons and its integration value is
12.29.
B = [12.29/3] = 4.09
(2)
(3)
DP = [A] + [B]
DP = 54.18 + 4.09
DP = 58.27 ∼ 58.
2.1.5.3. Deprotection of the Silyl Group of the Monohydroxyl-
Terminated Polybutadiene Polymer. In a 250 mL three-neck round-
bottom flask equipped with a Teflon-coated stir bar, a monohydroxyl-
terminated polybutadiene polymer (15 g) and tetrahydrofuran (150
mL) were added. Concentrated HCl (7 mL) was added dropwise to
the above solution under stirring. The reaction was continued for the
next 2 h. The reaction mixture was concentrated to 30%, and the
polymer was isolated by precipitation in MeOH. Precipitation was
repeated for two times and MeOH was removed by decantation. The
polymer was dried under vacuum for 3 h.
2.1.5. Synthesis of HTPB. HTPB was prepared by following a
modification of a process reported earlier.42 Detailed reaction steps
are described below.
1H NMR (CDCl3) δ (ppm) (Figure S5): 1.2−2.4 (−CH2 of a
linear chain proton), 3.4−3.6 (−OCH2, 4H), 4.9−5.1 (−CHCH2
of a vinyl proton), and 5.25−5.75 (−CHCH− of a linear chain
proton).
2.1.6. Synthesis of the Telechelic Br−PB−Br Macroinitiator. The
Br−PB−Br macroinitiator was prepared by modification of a process
2.1.5.1. Synthesis of a Silyl-Protected Hydroxyl Group Contain-
ing a Lithium Initiator. Step I Protection of the Hydroxyl Group of 3-
Chloro-propan-1-ol
To a solution of 3-chloro-propan-1-ol (5.1 mL, 60 mmol) in 100
mL of dichloromethane, imidazole (5.3 g, 78 mmol) was added and
stirred for 10 min. A solution of t-butyldimethylsilyl chloride (10.85 g,
C
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a
Table 1. Reaction Conditions and Molecular Characterization Data of PHisMAM-b-PB-b-PHisMAM Triblock Copolymers
b
c
d
d
e
entry
polymer
[HisMAM]0/[Br−PB−Br]0 conv. (%) Mn,theo (g mol−1
)
Mn,SEC (g mol−1
)
Đ
TCP (°C)
P1
P2
P3
P4
PHisMAM50-b-PB58-b- PHisMAM50
PHisMAM100-b-PB58-b- PHisMAM100
PHisMAM150-b-PB58-b- PHisMAM150
PHisMAM200-b-PB58-b- PHisMAM200
100: 58
200: 58
300: 58
400: 58
71
63
65
60
19,300
31,600
46,900
57,000
18,600
32,200
44,900
55,300
1.20
1.20
1.18
1.19
31
37
42
49
a
Reaction conditions: solvent = THF/H2O (1: 5); catalyst = Ni−Co alloy; ligand = Me6TREN; temperature = 25 °C; and time = 24 h.
b
c
Determined gravimetrically based on the monomer feed. Calculated using yield as conversion and the following equation: Mn,theo
=
([HisMAM]0/[Br−PB−Br]0 × yield × MHisMAM) + MBr−PB−Br, where MHisMAM (= 223 g mol−1) and MBr−PB−Br (= 3500 g mol−1) are the molecular
d
e
weights of HisMAM and Br−PB−Br, respectively. Obtained from SEC measurements. TCP measured by turbidimetry.
reported earlier.43 HTPB (1.37 g, 0.448 mmol) and triethylamine
4.6 −CH(COOH)CH2 (signal c′)
∫
(147 μL, 1.056 mmol) were dissolved in anhydrous THF (38 mL) in
a round-bottom flask under a N2 atmosphere, and the mixture was
allowed to cool down to 0 °C. Then, BiBB (120 μL, 0.968 mmol) was
added dropwise to that reaction mixture under a N2 atmosphere at 0
°C. The reaction mixture was stirred magnetically at 0 °C for 2 h and
then at an ambient temperature for another 24 h. After this, the
reaction mixture was filtered to remove the present triethylammonium
bromide salt and the filtrate was then evaporated using a rotary
evaporator to concentrate the reaction mixture. The concentrated
solution was then precipitated in chilled MeOH. The precipitate was
then dried under vacuum for 6 h at 40 °C to obtain the product,
which was characterized by NMR spectroscopy.
4.3
DP
=
× DP
n,Br−PB−Br
n,M
1
2
×
4.0 −CH2 (signal g)
∫
3.6
(4)
2.2. pH Titration. An aqueous solution of the copolymer (0.1 M)
was prepared using deionized water. The pH of the solution was
adjusted to pH 1.2 using HCl. This solution was titrated against 0.25
N NaOH solution until the pH reached 12.6. During the titration, the
change of pH of the solution was monitored using a pH meter.
2.3. Antifouling Study. Typically, 1.5 mL of aqueous solution of
the polymer (1 mg mL−1) was mixed with 1.5 mL of aqueous BSA
solution (1 mg mL−1) of pH 7 at 25 °C in a cuvette. The evolution of
the hydrodynamic diameter of the solution with time was monitored
using dynamic light scattering (DLS).
1H NMR (CDCl3) δ (ppm) (Figure 2a): 1.22 (methylene protons
in −CH2−CH−CHCH2), 1.39 (methyne signals in −CH2−CH−
CHCH2), 1.99 (methylene signals in −CH2−CHCH−CH2−
repeating units and methyl protons −(CH3)2C(Br) in bromo-initiator
residues), 3.10 (−CH2, signal g), 5.38 (methyne proton signals
−CHCH− in the cis/trans-1,4 olefinic structure), and 5.54 (alkenyl
proton signals −CHCH2 in the 1,2-vinyl-terminated structure).
2.1.7. Syntheses of PHisMAM-b-PB-b-PHisMAM Triblock Copoly-
mers. The syntheses of PHisMAM-b-PB-b-PHisMAM triblock
copolymers were carried out using Br−PB−Br as the macroinitiator
under a N2 atmosphere using Schlenk techniques. Typically, the Br−
PB−Br macroinitiator, Ni−Co nanoparticles, Me6TREN, and
degassed THF were taken in a flask under a nitrogen flow and
stirred magnetically and degassed by N2 bubbling for 20 min. Then,
degassed HisMAM solution in water was introduced into the flask
under a nitrogen flow, and the reaction mixture was heated at 25 °C
under constant stirring for 24 h. The polymerization reaction was
stopped at different time intervals to estimate the monomer
conversion (by gravimetry) and the molar mass (Mns) and dispersity
values (Đs) (by size-exclusion chromatography (SEC)). After
completion of the reaction, the crude product was dissolved in
water and the Ni−Co alloy catalyst was separated simply by holding a
magnetic bar at the wall of the flask. The crude product was then
precipitated in prechilled MeOH (×3 times) and filtered. The
precipitate was isolated and dried under vacuum for 6 h at 40 °C and
characterized by NMR spectroscopy and SEC.
2.4. Characterization. 2.4.1. NMR Spectroscopy. lH NMR
spectra of the synthesized polymers were recorded at 25 C using a
Bruker 400 MHz spectrometer.
ο
2.4.2. Attenuated Total Reflection Infrared (ATR-IR) Spectrosco-
py. The spectra were recorded using a PerkinElmer Spectrum 100 in
the ATR mode with a diamond crystal using 16 scans per spectrum
and a resolution of 4 cm−1 and a spectral range of 4000−600 cm−1.
2.4.3. Size Exclusion Chromatography (SEC). Mn and Đ values of
the polymers were determined using a SEC with a triple-detection
GPC (from Agilent Technologies) using a PL0390-0605390 LC light
scattering detector capable of detecting two diffusion angles (15 and
90°), a PL0390−06034 capillary viscometer, a 390-LC PL0390-0601
refractive index detector, and two PL1113-6300 ResiPore 300 mm ×
7.5 mm columns. DMF (containing 0.1 wt % of LiCl) was used as the
eluent at a flow rate of 0.8 mL min−1. The entire SEC-HPLC system
was thermostated at 35 °C, and narrow linear poly(methyl
methacrylate) standards were used to calibrate the SEC instrument.
The results were processed using the corresponding Agilent software.
2.5. UV−vis Spectroscopy. The thermosensitivity of the
PHisMAM-b-PB-b-PHisMAM copolymer solution was measured
using a Shimadzu UV-2600 spectrophotometer equipped with a
temperature-controlled sample holder. A 1.0 wt % solution of
PHisMAM-b-PB-b-PHisMAM in water was taken in a 3 mL quartz
cuvette and placed in a UV−vis spectrophotometer equipped with a
temperature controller. The transmittance of the copolymer solution
was monitored at a detection wavelength of 500 nm at a heating rate
of 1 °C min−1 in the temperature range of 10 to 80 °C.
1H NMR (D2O) δ (ppm) (Figure 2b, P1, Table 1): 1.08
(−CH2C(CH3) of the HisMAM repeating unit), 1.22 (methylene
protons in −CH2−CH−CHCH2), 1.40 (−CH2C(CH3) of the
HisMAM repeating unit), 1.99 (methylene signals in −CH2−CH
CH−CH2− repeating units and methyl protons −(CH3)2C(Br) in
bromo-initiator residues), 3.20 (−CH2CH(COOH) of the HisMAM
repeating unit), 4.60 (−CH(COOH)CH2 of the HisMAM repeating
unit), 3.80 (−CH2, signal g), 5.38 (methyne proton signals −CH
CH− in the cis/trans-1,4 olefinic structure), 5.54 (methyne proton
signals −CHCH2 in the 1,2-vinyl-terminated structure), 7.28
(−NH of the HisMAM repeating unit), and 7.32−7.52 (5H of the
aromatic ring of the HisMAM repeating unit).
2.6. Dynamic Light Scattering (DLS). The hydrodynamic
diameter (Dh) of the aqueous solutions of the copolymers was
measured using a Malvern particle size analyzer (Zetasizer NANO
ZS90).
3. RESULTS AND DISCUSSION
3.1. Synthesis of PHisMAM-b-PB-b-PHisMAM Triblock
Copolymer Biohybrids. The L-histidine-derived monomer,
HisMAM, was prepared via a simple reaction between L-
histidine with methacryloyl chloride (see Experimental Section
for the detailed procedure, Scheme S1) and characterized by
IR and NMR spectroscopies (see Supporting Information,
Figures S1,S2). The telechelic dibromo-terminated polybuta-
Degree of polymerization (DP) calculation
For the PHisMAM-b-PB-b-PHisMAM (P1−P4, Table 1), using 1H
NMR spectroscopy,
D
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diene (Br−PB−Br) macroinitiator was prepared via living
anionic polymerization of butadiene, followed by end capping
and postpolymerization modification (Scheme 1) and
characterized by NMR spectroscopy (see Supporting In-
formation, Figures S3−S5) and SEC (Figure 1). The 1H NMR
confirms the product structure. The H NMR spectrum of
PHisMAM-b-PB-b-PHisMAM (Figure 2b) reveals emergence
of new signals at 1.08 (−CH2C(CH3) of the HisMAM
repeating unit), 1.40 (−CH2C(CH3) of the HisMAM
repeating unit), 3.20 (−CH2CH(COOH) of the HisMAM
repeating unit), 4.60 (−CH(COOH)CH2 of the HisMAM
repeating unit), 7.28 (−NH of the HisMAM repeating unit),
and 7.32−7.52 (5H of the aromatic ring of the HisMAM
repeating unit).45
Kinetic studies suggested the controlled nature of the
polymerization, as proven by the pseudo-first-order kinetic plot
and linear evolution of Mn with monomer conversion, retaining
low Đ values (Figure 3). From the slope of Figure 3a, the
apparent propagation rate constant kp(app) was determined to
be 1.6 × 10−5 s−1.
Recyclability of the catalyst is an important characteristic of
heterogeneous catalysis. Thus, to prove the ease of catalyst
recyclability, the alloy catalyst was isolated just by using a bar
magnet after completion of the polymerization and used for
the next polymerization reaction. Results (Table S1) revealed
that there is not any appreciable reduction in the catalyst
efficiency (both pertaining to the control of the RDRP process
and the molecular weight parameters of the prepared triblock
copolymers) even after four polymerization cycles.
3.2. pH and Temperature Response of PHisMAM-b-
PB-b-PHisMAM Triblock Copolymer Biohybrids. The pH
response of the copolymer (Figure 4) revealed that the L-
histidine-derived copolymer, PHisMAM-b-PB-b-PHisMAM,
existed in three different charged states27 depending on the
pH of the solution. At low pH conditions (pH < 1.8), both
carboxylate and imidazole groups were protonated, leading to a
monocationic state of the PHisMAM-b-PB-b-PHisMAM
copolymer. At high pH values (pH > 7.1), both the carboxylate
and imidazole groups were deprotonated and the copolymer
existed in its monoanionic state. At an intermediate pH range
(between pH 1.8 and 7.1), the carboxylate groups were
Figure 1. Evolution of SEC traces of the Br−PB−Br macroinitiator
and PHisMAM-b-PB-b-PHisMAM triblock copolymers (P1−P4,
Table 1).
spectrum of Br−PB−Br (Figure 2a) shows characteristic
signals at 1.22 (methylene protons in −CH2−CH−CH
CH2), 1.99 (methylene signals in −CH2−CHCH−CH2−
repeating units and methyl protons −(CH3)2C(Br) in bromo-
initiator residues), 5.38 (methyne proton signals −CHCH−
in the cis/trans-1,4 olefinic structure), and 5.54 (alkenyl proton
signals −CHCH2 in the 1,2-vinyl-terminated structure).44
A range of PHisMAM-b-PB-b-PHisMAM triblock copoly-
mer syntheses (P1−P4, Table 1) was carried out via recyclable
alloy-mediated RDRP using Br−PB−Br as the macroinitiator
(Scheme 1). The SEC analysis revealed successful synthesis of
the well-defined PHisMAM-b-PB-b-PHisMAM triblock co-
polymers (Figure 1), showing little or no starting Br−PB−Br
macroinitiator. The presence of characteristic signals of the
PB43,44 central block and the PHisMAM45 terminal block in
the 1H NMR (Figure 2b) and IR spectrum (Figure S6)
1
Figure 2. H NMR spectra for (a) Br−PB−Br (in CDCl3) and PHisMAM-b-PB-b-PHisMAM (P1, Table 1) (in D2O).
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Figure 3. Plot of (a) conversion versus time, ln([M]0/[M]) versus time, and (b) evolution of Mn and Đ with increasing monomer conversion for
alloy-mediated RDRP of HisMAM in THF/H2O 1:5 at 25 °C using Br−PB−Br as the initiator.
thermal energy is present to break the polymer−water H-
bonds. Subsequently, the copolymer forms H-bonds with itself
via amide and −COOH functionalities, resulting in a coil to
globule transition. With the increase of the HisMAM mol % in
the copolymer, the LCST increases as there are more amide
functionalities present to form H-bonds with water molecules,
making the copolymer soluble until sufficient thermal energy is
provided.
The effect of pH on the LCST of the copolymer was
measured using PHisMAM50-b-PB58-b-PHisMAM50 as the
representative sample. At pH 7.0, the LCST of the copolymer
(P1, Table 1) was approximately 31 °C. When pH of the
solution was adjusted to 1.5 and 10.5, in both cases, the LCST
increased to 35 and 41 °C, respectively. This is probably due to
the fact that the copolymer in its cationic (at pH 1.5) or
anionic (at pH 10.5) state has higher solubility in water due to
the enhanced hydrophilicity of the polymer chain compared to
its zwitterionic state (at pH 7.0).
3.3. Antifouling Behavior of PHisMAM-b-PB-b-PHis-
MAM Triblock Copolymer Biohybrids. The pH titration
(Figure 4) revealed that the PHisMAM-b-PB-b-PHisMAM
copolymer existed in its zwitterionic form in the pH ranges of
1.8−7.1. Zwitterionic polymers in aqueous solution have the
ability to strongly bind to H2O molecules via electrostatically
induced hydration and thus demonstrate antifouling perform-
ance.46 Compared to PEG,29 the most widely used antifouling
material, which is nonbiodegradable and leads to bioaccumu-
lation,30 zwitterionic polymers offer a broader chemical
diversity and greater freedom for molecular design and may
emerge as a degradable, nonbioaccumulate alterative to PEG
for the development of antifouling materials suitable for
biomedical applications.47
Figure 4. pH titration curve for the PHisMAM-b-PB-b-PHisMAM
copolymer for a pH range of 1.2 to 12.6.
deprotonated and the imidazole groups were protonated,
leading to the zwitterionic state of the copolymer. These pH-
tunable charged states of the copolymer can be utilized for
antifouling and other physical properties. Notably, throughout
all pH ranges, the copolymers were not observed to aggregate
and remained as unimers, as revealed by a Dh of <5 nm.
The synthesized amphiphilic PHisMAM-b-PB-b-PHisMAM
triblock copolymers exhibited an unprecedented dual temper-
ature response, whereas neither HTPB nor PHisMAM was
thermoresponsive. The cloud point (TCP) of the PHisMAM-b-
PB-b-PHisMAM copolymers, as determined by turbidimetry,
increased with an increase of hydrophilic PHisMAM segments
in the copolymer (Table 1 and Figure 5).
The origin and variation of TCP as a function of the
HisMAM content in the copolymer might be explained as
follows. Below the lower critical solution temperature (LCST),
water molecules form H-bonds with the pendent amide
functionalities of the copolymer, giving it a coil conformation,
which remains soluble in water. Above the LCST, enough
In order to investigate the antifouling nature in solutions, the
resistivity of BSA, as a representative protein, was tested
Figure 5. TCP as a function of the HisMAM content in PHisMAM-b-PB-b-PHisMAM triblock copolymers at pH 7.0.
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toward the PHisMAM-b-PB-b-PHisMAM copolymer and the
result was compared with a representative fouling polymer
(PNIPAM)48 and an antifouling polymer (PEG) (see
Experimental Section for more details). The Dh of the solution
mixture containing the polymer (PHisMAM-b-PB-b-PHis-
MAM or PNIPAM or PEG) and BSA was estimated by DLS
at different time intervals at pH 7 with the assumption that a
fouling polymer will allow aggregation of the polymer leading
to an increase of the Dh while an antifouling polymer will not
allow any fouling and the Dh will remain the same with time.
The results (Figure 6) revealed that Dh of the solution
catalyst synthesis, handling, recovery, reusability, and protect-
ing group-free chemistry may allow wide applicability of this
facile synthesis protocol for the synthesis of biohybrids for
application in some promising areas including biomedical
science and materials science. The synthesized multistimuli-
responsive antifouling biohybrid may be useful for the
construction of a multistimuli-responsive actuator for bio-
medical applications.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biomac.1c00748.
Catalyst recyclability study, synthesis of HisMAM, and
ATR-IR and NMR spectra of compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Sanjib Banerjee − Department of Chemistry, Indian Institute
of Technology Bhilai, Raipur 492015 Chhattisgarh, India;
orcid.org/0000-0003-4841-4408;
Email: sanjib.banerjee@iitbhilai.ac.in
Figure 6. Variation of the hydrodynamic diameters of the aqueous
solutions of PNIPAM, PEG, and PHisMAM-b-PB-b-PHisMAM in the
presence of BSA at 25 °C.
Authors
Sk Arif Mohammad − Department of Chemistry, Indian
Institute of Technology Bhilai, Raipur 492015 Chhattisgarh,
India; orcid.org/0000-0002-2162-4416
increased for PNIPAM, indicating fouling and formation of
aggregates via hydrophobic interactions of BSA with PNIPAM.
However, when PEG or PHisMAM-b-PB-b-PHisMAM was
used, the Dh value remained the same even after 48 h,
suggesting the absence of any fouling. This result indicated that
PHisMAM-b-PB-b-PHisMAM offers significant resistance
toward protein adsorption and may be useful for the
preparation of an antifouling surface and employed as an
alternative to PEG. Notably, all the copolymers (P1−P4, Table
1) exhibited an antifouling behavior only at their zwitterionic
state (at pH 7). None of these copolymers exhibited the
antifouling behavior in their cationic form. This is in
accordance with the other amino acid-derived polymers that
exhibited the antifouling behavior only at their zwitterionic
state.31−34 The copolymer exhibited the antifouling behavior
even when higher concentrations of BSA (3 and 5 mg mL−1)
were used. Notably, control samples, poly(histidine meth-
acrylamide) (PHisMAM) and poly(lysine methacrylamide)
(PLysMAM), in their zwitterionic state, exhibited an
antifouling behavior against BSA. The Dh value remained the
same as that at time t = 0 even after 48 h.
Subrata Dolui − Department of Chemistry, Indian Institute of
Technology Bhilai, Raipur 492015 Chhattisgarh, India
Devendra Kumar − Department of Chemistry, Indian Institute
of Technology Bhilai, Raipur 492015 Chhattisgarh, India;
orcid.org/0000-0003-0354-1489
Shivshankar R. Mane − Polymer Science and Engineering
Division, CSIR-National Chemical Laboratory, Pune,
Maharashtra 411008, India; orcid.org/0000-0003-3853-
449X
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biomac.1c00748
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We appreciate financial support from the Science and
Engineering Research Board, India through the Ramanujan
Fellowship Award (SB/S2/RJN-113/2016) and the Early
Career Research Award (ECR/2018/001990) and IIT Bhilai
for the Research Initiation Grant. S.A.M. and D.K. thank the
MHRD, Govt. of India and CSIR, Govt. of India for their
fellowships, respectively. S.R.M. thank the CSIR-SRA
(Scientist’s Pool Scheme, Award No. 13(9139-A)/2020-
Pool) for the funding. We would like to thank Prof. Tarun
K. Mandal (Indian Association for the Cultivation of Science),
Dr. P. P. Wadgaonkar (CSIR-NCL), and Dr. A. V. Ambade
(CSIR-NCL) for fruitful discussions.
4. CONCLUSIONS
In summary, we report, for the very first time, synthesis of a
novel amphiphilic PHisMAM-b-PB-b-PHisMAM triblock
copolymer composed of L-histidine (ampholyte) and butadiene
(hydrophobic moiety) via mechanistic transformation from
living anionic polymerization into recyclable alloy-mediated
RDRP. This new class of novel biohybrid allows the
integration of functionalities of two different blocks (a
synthetic polymer and a biomolecule). The synthesized
zwitterionic copolymer is capable of switching among
zwitterionic, anionic, and cationic forms, exhibiting dual (pH
and temperature) stimuli-responsive properties and reducing
adsorption of proteinse. The collective advantages of ease of
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