Construction of polymer-protein bioconjugates with varying chain topologies: Polymer molecular weight and steric hindrance effects

Article abstract of DOI:10.1002/asia.201100489

We report on the fabrication of well-defined polymer-protein bioconjugates with varying chain architectures, including star polymers, star block copolymers, and heteroarm star copolymers through the specific noncovalent interaction between avidin and biotinylated synthetic polymer precursors. Homopolymer and diblock precursors site-specifically labeled with a single biotin moiety at the chain terminal, chain middle, or diblock junction point were synthesized by a combination of atom-transfer radical polymerization (ATRP) and click reactions. By taking advantage of molecular recognition between avidin and biotin moieties, supramolecular star polymers, star block copolymers, and heteroarm star copolymers were successfully fabricated. This specific binding process was also assessed by using the diffraction optic technology (DOT) technique. We further investigated the effects of polymer molecular weights, location of biotin functionality within the polymer chain, and polymer chain conformations, that is, steric hindrance effects, on the binding numbers of biotinylated polymer chains per avidin within the polymer-protein bioconjugates, which were determined by the standard avidin/2-(4- hydroxyazobenzene)benzoic acid (HABA) assay. The binding numbers vary in the range of 1.9-3.3, depending on the molecular weights, locations of biotin functionality within synthetic polymer precursors, and polymer chain conformations.

Full text of DOI:10.1002/asia.201100489

DOI: 10.1002/asia.201100489
Construction of Polymer–Protein Bioconjugates with Varying Chain
Topologies: Polymer Molecular Weight and Steric Hindrance Effects
Xuejuan Wan,[a] Guoying Zhang,*[a] Zhishen Ge,[a] Ravin Narain,*[b] and Shiyong Liu*[a]
On the occasion of the 10th anniversary of click chemistry
Abstract: We report on the fabrication
of well-defined polymer–protein bio-
conjugates with varying chain architec-
tures, including star polymers, star
block copolymers, and heteroarm star
copolymers through the specific nonco-
valent interaction between avidin and
biotinylated synthetic polymer precur-
sors. Homopolymer and diblock pre-
cursors site-specifically labeled with a
single biotin moiety at the chain termi-
nal, chain middle, or diblock junction
point were synthesized by a combina-
tion of atom-transfer radical polymeri-
zation (ATRP) and click reactions. By
taking advantage of molecular recogni-
tion between avidin and biotin moiet-
ies, supramolecular star polymers, star
block copolymers, and heteroarm star
copolymers were successfully fabricat-
ed. This specific binding process was
also assessed by using the diffraction
optic technology (DOT) technique. We
further investigated the effects of poly-
mer molecular weights, location of
biotin functionality within the polymer
chain, and polymer chain conforma-
tions, that is, steric hindrance effects,
on the binding numbers of biotinylated
polymer chains per avidin within the
polymer–protein bioconjugates, which
were determined by the standard
avidin/2-(4-hydroxyazobenzene)benzoic
acid (HABA) assay. The binding num-
bers vary in the range of 1.9–3.3, de-
pending on the molecular weights, lo-
cations of biotin functionality within
synthetic polymer precursors, and poly-
mer chain conformations.
Keywords: bioconjugates
chemistry polymers
·
click
steric
·
·
hindrance · supramolecular chemis-
try
Introduction
biotechnology fields, such as delivery of protein and gene
therapeutics, enzyme catalysis, tissue engineering, bionanor-
eactors, and artificial enzymes or cells.[1] Concerning their
synthesis, two main types of design strategies have been de-
veloped. The conventional one relies on the covalent or
noncovalent conjugation of synthetic polymer precursors
onto biomacromolecules, such as proteins. In the covalent
approach, quantitative, highly efficient coupling reactions,
such as amidation reactions between activated esters and
amine functionalities,[2] thiol–ene[3] and azide–alkyne 1,3-di-
polar cycloaddition click reactions,[4] and thiol/pyridyl disul-
fide exchange reactions,[5] have been frequently utilized to
fabricate well-defined hybrid bioconjugates. As for the non-
covalent approach, biotin/avidin,[6] cofactor/apoprotein re-
constitution,[7] saccharide/concanavalin A,[8] and other spe-
cific host–guest molecular recognition processes[9] have been
employed. The second type of strategy relies on the use of
well-developed controlled/living polymerization tech-
niques,[10] such as atom-transfer radical polymerization
(ATRP) and reversible addition–fragmentation chain trans-
fer (RAFT) polymerization, which allow for the direct prep-
aration of well-defined polymer–protein conjugates from
protein-based ATRP initiators[11] and macroRAFT agents.[12]
The covalent or noncovalent attachment of synthetic poly-
mer chains onto proteins or other biomacromolecules can
endow them with improved solubility and stability, increased
plasma half-lives and bioavailability, and reduced immuno-
genicity. Biohybrid polymers and their self-assembled nano-
structures have found broad applications in biomedical and
[a] Dr. X. Wan, Prof. G. Zhang, Prof. Z. Ge, Prof. S. Liu
CAS Key Laboratory of Soft Matter Chemistry
Hefei National Laboratory for Physical Sciences at the Microscale
Department of Polymer Science and Engineering
University of Science and Technology of China
Hefei, Anhui 230026 (P.R. China)
Fax : (+86) 551-360-7348
E-mail: sliu@ustc.edu.cn
gyzhang@ustc.edu.cn
[b] Prof. R. Narain
Department of Chemical and Materials Engineering
University of Alberta
Edmonton, Alberta T6G 2G6 (Canada)
Fax : (+1) 780-492-2881
E-mail: narain@ualberta.ca
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201100489.
Chem. Asian J. 2011, 6, 2835 – 2845
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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FULL PAPERS
Of the above approaches, the strong, specific, noncovalent
interaction between avidin and biotin, upon appropriate
functionalization with a synthetic polymer chain segment,
was often employed to construct supramolecular synthetic
polymer–protein hybrid bioconjugates.[13] Compared with
the covalent approach, noncovalent ones are less disruptive
to the native protein conformation and relevant biological
activities. Avidin contains four binding sites for biotin, and
the biotin/avidin pair possesses a high binding affinity with
Kd values up to 10ꢀ15 m. However, it has been reported that
biotinylated polymers, depending on their molecular weight
(MW) and chain topology, typically possess decreased affini-
ty for avidin. Previously, Maynard et al. reported the fabri-
cation of polymer/avidin bioconjugates by either direct
ATRP from avidin-based initiators or noncovalent conjuga-
tion between avidin and biotinylated poly(N-isopropylacry-
lamide) (PNIPAM).[14] In the latter case, the binding
number of PNIPAM chain per avidin was quantified to be
3.6; thus, supramolecular star polymers possessing an avidin
core and about three to four PNIPAM arms were prepared.
Haddleton et al. and Frey et al. fabricated poly(amidoa-
mine) (PAMAM) dendrimer and linear-dendritic diblock co-
polymers with a biotin functionality attached at the dendri-
mer focal point or the linear chain terminal, respectively.[15]
Their subsequent conjugation with avidin afforded star poly-
mer bioconjugates, as evidenced by the standard 2-(4-hy-
droxyazobenzene)benzoic acid (HABA)/avidin assay. In
both cases, the actual binding numbers of dendritic or
linear-dendritic arms per avidin were not discussed. In 1997,
Gruber et al. reported the conjugation of biotinylated poly-
ethylene glycol (PEG) with avidin, and the effects of PEG
chain length and end-group modification with hydrophobic
dyes or other functionalities on the time-dependent structur-
al evolution of as-prepared bioconjugates.[16] Recently,
Kwon et al. reported that the binding numbers of biotinylat-
ed PEG/avidin conjugates are highly dependent on the PEG
chain length, which decreased from about four to one when
the PEG MW increased from 588 to 5000 Da.[17]
polymers and heteroarm star copolymers, can also be fabri-
cated.
Herein, we report on the fabrication of well-defined poly-
mer–protein bioconjugates with varying chain architectures,
including star polymers, star block copolymers, and hetero-
arm star copolymers, through the specific noncovalent inter-
action between avidin and biotinylated synthetic polymer
precursors. Homopolymer and diblock precursors site-spe-
cifically labeled with one single biotin moiety at the chain
terminal, chain middle, or diblock junction point were syn-
thesized by a combination of ATRP and click reactions
(Scheme 1). By taking advantage of the specific molecular
Scheme 1. Schematic illustration of a) the synthesis of homopolymers and
diblock copolymers with one single alkynyl functionality at different loca-
tions (chain terminal, chain middle, and diblock junction point), and
b) click synthesis of water-soluble thermoresponsive homopolymers and
diblock copolymers site-specifically labeled with biotin functionality.
PDMA=poly(N,N-dimethylacrylamide).
In addition to the above-described MW effects of biotiny-
lated polymers on their noncovalent conjugation with
avidin, the location of biotin functionality within the poly-
mer precursor (chain terminal or chain middle) might also
play crucial roles. Based on chemical intuition, precursors
with the biotin moiety attached at the chain middle should
suffer from increased steric hindrance during their conjuga-
tion with avidin. However, this aspect has been far less ex-
plored and no quantitative data were presented in relevant
literature reports. It is thus highly desirable to quantitatively
determine the binding numbers and systematically investi-
gate the effects of MW and chemical structures of biotinylat-
ed polymers on the formation of hybrid bioconjugates.
Moreover, previous reports in this area have focused on the
fabrication of synthetic polymer/avidin bioconjugates with a
star-type topology by employing biotin-terminated linear
polymers and dendrimers. By appropriately designing the
chemical structures of biotinylated polymers, hybrid biocon-
jugates with other chain topologies, such as star block co-
recognition between avidin and biotin moieties, supramolec-
ular star polymers, star block copolymers, and heteroarm
star copolymers were successfully fabricated (Scheme 2).
Most importantly, the effects of polymer MW and the loca-
tion of the biotin functionality within polymer chains, that
is, steric hindrance effects, on their binding affinity with
avidin and the binding numbers (e.g., number of noncova-
lently grafted polymer arms) were further quantitatively de-
termined by the standard avidin/HABA assay.
Experimental Section
Materials
N-Isopropylacrylamide (NIPAM; TCI) was recrystallized twice from ben-
zene/hexane (2:1 v/v) prior to use. N,N-Dimethylacrylamide (DMA;
TCI) was vacuum distilled from calcium hydride and stored at ꢀ158C
prior to use. 1,1’-Carbonyldiimidazole (CDI), N,N,N’,N’’,N’’-pentamethyl-
diethylenetriamine (PMDETA), 2-chloropropionyl chloride, CuCl, CuBr,
2836
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 2835 – 2845

Construction of Polymer–Protein Bioconjugates
Biotin (0.61 g, 2.5 mmol) was first dis-
solved in N,N-dimethylformamide
(DMF; 15 mL) at 558C. After cooling
to room temperature, CDI (0.73 g,
4.5 mmol) in DMF (3 mL) was slowly
added and the reaction mixture was al-
lowed to stir for 3 h at room tempera-
ture. Subsequently, 1-azido-3-amino-
propane (0.75 g, 7.5 mmol) in DMF
(6 mL) was added dropwise over
about 0.5 h and the reaction mixture
was left stirring for another 12 h. The
solvent was then removed in vacuo
and the residues were purified by re-
crystallization in a mixture of 1-buta-
nol/acetic acid/water (70:7:10, v/v/v).
The obtained white solid was washed
with diethyl ether three times and
dried in
a vacuum oven to afford
biotin azide (0.74 g, 90.7%). 1H NMR
(CD3OD, Figure 1a): d=4.50 (m, 1H;
ꢀ ꢀ ꢀ ꢀ
ꢀ
ꢀ
S CH2 CH ), 4.33 (m, 1H;
S
CH2
ꢀ
ꢀ
ꢀ
CH CH ), 3.38 (m, 4H;
ꢀ ꢀ
ꢀ
CH2CH2N3), 3.22 (m, 1H; S CH
CH ), 2.93, 2.72 (t, 2H; S CH2
CH ), 2.19 (t, 2H; CH2CONHCH2),
1.54–1.83 (m, 6H;
ꢀ
ꢀ ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
CH2CH2CH2
ꢀ
ꢀ
CH2CO ), 1.44 ppm (m, 2H; CH2
CH2CH2N3).
Synthesis of Alkynyl-PNIPAM,
Alkynyl-PNIPAM-b-PDMA, and
Alkynyl-(PNIPAM)2 (Scheme 3b and
c)
Scheme 2. Schematic illustration of the fabrication of polymer–protein bioconjugates with varying chain topol-
ogies (star polymers, star block copolymers, and heteroarm star copolymers) through specific noncovalent in-
teractions between avidin and synthetic homopolymer and diblock copolymers site-specifically labeled with
one single biotin functionality.
Alkynyl-PNIPAM, alkynyl-PNIPAM-
b-PDMA, and alkynyl-(PNIPAM)2
were prepared by the ATRP or con-
secutive ATRP techniques in isopro-
2,2-bis(hydroxymethyl)propionic acid (bis-MPA), propargyl alcohol,
biotin, and avidin were purchased from Aldrich and used as received. All
other reagents were purchased from Shanghai Chemical Reagent Co. and
used as received. HABA/avidin reagent (lyophilized powder) was pur-
chased from Sigma and used according to standard procedures. 1-Azido-
3-aminopropane,[18] tris[2-(dimethylamino)ethyl]amine (Me6TREN),[19] al-
kynyl-functionalized resin (alkynyl-resin),[20] azide-terminated poly(ethy-
lene oxide) monomethyl ether (PEO-N3),[21] ATRP initiators, including
propargyl 2-chloropropionate[22] and alkynyl-Cl2,[23] and methyl 3,5-bis(-
propargyloxy)benzyl alcohol (alkynyl-OH-alkynyl),[20] were synthesized
according to literature procedures (Scheme 3). Water was deionized with
a Milli-Q SP reagent water system (Millipore) to a specific resistivity of
panol using propargyl 2-chloropropionate (for alkynyl-PNIPAM and al-
kynyl-PNIPAM-b-PDMA diblock) and alkynyl-Cl2 (for alkynyl-
(PNIPAM)2) as the initiators, according to well-established literature pro-
cedures.[24] Structural parameters were characterized by 1H NMR spec-
troscopy and gel permeation chromatography (GPC) and the results are
listed in Table 1.
Synthesis of PEO-Alkynyl-PNIPAM Diblock Copolymer Bearing One
Alkynyl Functionality at the Junction Point (Scheme 3d)
PEO-OH-alkynyl was prepared by the click reaction of PEO-N3 with an
excess of alkynyl-OH-alkynyl. PEO45-N3 (1.0 g, 0.5 mmol N3 moieties), al-
kynyl-OH-alkynyl (2.16 g, 10 mmol), and PMDETA (0.1 g, 0.6 mmol)
were dissolved in DMF (100 mL) in a Schlenk flask. The flask was sub-
jected to three freeze/pump/thaw cycles and CuBr (72 mg, 0.5 mmol) was
then introduced under the protection of an N2 atmosphere. After one ad-
18.4 MWcmꢀ1
.
Sample Preparation
General approaches employed for the
synthesis of biotinylated polymer pre-
cursors and polymer–protein bioconju-
gates with varying chain architectures
(star polymers, star block copolymers,
and heteroarm star polymers) through
specific noncovalent interactions be-
tween avidin and biotin moieties
within polymer precursors are shown
in Schemes 1–3.
Table 1. Structural parameters of alkynyl-functionalized polymer precursors with varying chain topologies, as
shown in Scheme 1.
[a]
[b]
[b]
Samples
Mn,NMR
Mn
Mw/Mn
DP[a]
PNIPAM
PDMA
PEO
alkynyl-PNIPAM19
alkynyl-PNIPAM50
2300
5800
2900
14400
6500
4400
7500
3100
6900
4200
13300
6200
6100
8300
1.07
1.08
1.09
1.09
1.07
1.07
1.10
19
50
19
69
27
15
45
–
–
8
65
–
–
–
–
–
–
45
45
alkynyl-PNIPAM19-PDMA8
alkynyl-PNIPAM69-PDMA65
PNIPAM27-alkynyl-PNIPAM27
PEO45-alkynyl-PNIPAM15
PEO45-alkynyl-PNIPAM45
Synthesis of Azide-Functionalized
Biotin (Biotin Azide) (Scheme 3a)
–
–
Biotin azide was synthesized by the
CDI-mediated esterification reaction.
[a] Determined by 1H NMR spectroscopy in CDCl3. DP=degree of polymerization. [b] Determined by GPC
using DMF as the eluent.
Chem. Asian J. 2011, 6, 2835 – 2845
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2837

G. Zhang, R. Narain, S. Liu et al.
FULL PAPERS
10 min and a solution of 2-chloropro-
pionyl chloride (51 mL, 0.5 mmol) in
anhydrous THF (5 mL) was then
added dropwise by a dropping funnel
over 30 min under vigorous stirring.
The reaction mixture was stirred in an
ice water bath for 1 h and then at
room temperature overnight. The in-
soluble salts were then filtered off and
all the solvents were removed by
rotary evaporation. The crude product
was dissolved in CH2Cl2 and precipi-
tated into excess diethyl ether. This
dissolution/precipitation cycle was re-
peated twice. After drying in
a
vacuum oven at room temperature to
give a constant weight, PEO-alkynyl-
Cl was obtained as a white powder
(0.45 g, 62.4% yield; Mn =2 300, Mw/
Mn =1.08).
PEO-alkynyl-PNIPAM was synthe-
sized by ATRP of NIPAM monomer
using PEO-alkynyl-Cl as the initiator
following well-established literature
procedures, and the structural parame-
ters of PEO-alkynyl-PNIPAM precur-
sors are summarized in Table 1.
Synthesis of Biotinylated Polymers
(Scheme 1)
Typical procedures are described as
follows: Biotin azide (0.06 mmol), al-
kynyl-functionalized polymer precur-
sor (0.05 mmol alkynyl moieties), and
PMDETA (0.06 mmol) were dissolved
in DMF (20 mL) in a Schlenk flask.
After the flask was subjected to three
freeze/pump/thaw
cycles,
CuBr
(0.06 mmol) was introduced under the
protection of an N2 atmosphere. The
reaction mixture was allowed to stir
overnight at room temperature under
the protection of N2. Alkynyl-function-
alized resin (70 mg, ꢁ0.1 mmol alkyn-
yl moieties) was added and the reac-
tion mixture was left stirring under the
protection of N2 for another 4 h. After
filtering off the resins, the filtrate was
evaporated to dryness under reduced
pressure. The residues were dissolved
in CH2Cl2 and passed through a neu-
tral alumina column to remove the
catalyst. The final product was dried in
Scheme 3. Schematic illustration of the synthesis of a) biotin azide and alkynyl-functionalized precursors: b) al-
kynyl-PNIPAM and alkynyl-PNIPAM-b-PDMA, c) PNIPAM-alkynyl-PNIPAM, and d) PEO-alkynyl-
PNIPAM. DMF=N,N-dimethylformamide, TEA=triethylamine, THF=tetrahydrofuran.
vacuo to give
a constant weight at
room temperature. The structural pa-
rameters of all biotinylated polymers (biotin-PNIPAM, biotin-PNIPAM-
b-PDMA, PNIPAM-biotin-PNIPAM, and PEO-biotin-PNIPAM) with
varying MW and chain architectures were characterized by 1H NMR
spectroscopy and GPC, and the results are shown in Table 2.
ditional freeze/pump/thaw cycle, the click reaction was carried out at
808C for 4 h under the protection of N2. After removing all of the sol-
vents under reduced pressure, the reaction mixture was then dissolved in
CH2Cl2 and passed through a neutral alumina column to remove the cat-
alyst. The elution was concentrated on a rotary evaporator and the crude
product was obtained by precipitation into an excess of ethyl ether. The
above dissolution/precipitation cycle was repeated three times. The col-
lected white solid was washed and dried in a vacuum oven at room tem-
perature to give a constant weight (0.82 g, 74.0% yield; Mn =2 200, Mw/
Mn =1.09).
HABA/Avidin Assay
The HABA/avidin reagent purchased from Sigma was reconstituted with
deionized water (10 mL) in a glass vial that contained 0.3 mm HABA,
avidin (0.45 gLꢀ1, 6.6ꢁ10ꢀ6 molLꢀ1), and buffering salts at pHꢁ7.3. In a
quartz cuvette containing 900 mL of the above-prepared HABA/avidin
assay solution, the optical absorbance at 500 nm (AHABA/avidin) was record-
ed. Then, an aqueous solution of biotinylated polymers (biotin-PNIPAM,
biotin-PNIPAM-b-PDMA, PNIPAM-biotin-PNIPAM, or PEO-biotin-
PEO-OH-alkynyl (0.60 g, 0.27 mmol OH functionality), TEA (50 mg,
0.5 mmol), and anhydrous THF (20 mL) were added to a 100 mL dry
round-bottomed flask. The flask was immersed in an ice water bath for
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Chem. Asian J. 2011, 6, 2835 – 2845

Construction of Polymer–Protein Bioconjugates
Table 2. Structural parameters of biotinylated polymers and average binding numbers of biotinylated polymer
chains per avidin.
this property has successfully
utilized for the construction of
polymer/avidin bioconjugates.[6]
However, the determination of
actual structural information of
as-prepared hybrid bioconju-
gates relies on the quantifica-
tion of binding numbers and
the understanding of effects of
polymer MW and location of
biotin moieties within the poly-
mer on the binding affinity. In
the current work, a series of
biotinylated homopolymers and
[a]
[b]
[b]
Samples
Mn
Mn
Mw/Mn
Binding numbers[c]
biotin-PNIPAM19
biotin-PNIPAM50
2600
6100
3200
14700
6800
4700
7800
3500
7400
4700
13800
6400
6300
8500
1.06
1.08
1.08
1.06
1.05
1.07
1.08
3.3
2.9
2.9
1.9
1.9
2.5
2.2
biotin-PNIPAM19-b-PDMA8
biotin-PNIPAM69-b-PDMA65
PNIPAM27-biotin-PNIPAM27
PEO45-biotin-PNIPAM15
PEO45-biotin-PNIPAM45
1
[a] Determined by H NMR spectroscopy in CDCl3 or [D4]MeOH. [b] Determined by GPC using DMF as the
eluent. [c] Binding numbers of biotinylated polymer chains per avidin as determined by the standard HABA/
avidin assay.
PNIPAM with varying chain lengths and block length ratios) at a prede-
termined concentration was added in a batch to the cuvette and the ab-
diblock copolymers with varying chain architecture and
chain lengths were synthesized, and the location of biotin
within the synthetic polymer chains ranges from the chain
terminal to the chain middle and diblock junction point
(Scheme 2). Taking advantage of the special interaction be-
tween avidin and the biotin moiety, we fabricated bioconju-
gates with varying star-type topologies, including star poly-
mers, star block copolymers, and heteroarm star polymers
(Scheme 3). We further quantified the binding numbers (i.e.,
number of grafted polymer chains per star-type bioconju-
gate) of biotinylated polymer chains by the standard
HABA/avidin assay and explored the effects of polymer
MW and biotin location within the polymer chain, that is,
steric hindrance effects, on the formation of polymer–pro-
tein hybrid bioconjugates.
sorbance at 500 nm (AHABA/avidin+sample) was then recorded. The binding
numbers of biotinylated polymer chains per avidin were calculated by
using Equations (1)–(2, and 3):
DA500nm ¼ aAHABA=avidinꢀAHABA=avidinþsample
ð1Þ
in which a is the dilution factor of HABA/avidin assay solution upon the
addition of aqueous solution of biotinylated polymer).
mmol biotin=mL ¼ ðDA500nm=34Þ=b
ð2Þ
in which the mm extinction coefficient at 500 nm is 34 and b is dilution
factor of biotinylated polymer aqueous solution after addition into the
cuvette).
mmol biotin=mL sample
binding number ðmole biotin=mole avidinÞ ¼
mmol avidin=mL sample
ð3Þ
Characterization
Synthesis of Biotinylated Polymers of Varying MWs and
Chain Architectures
1H NMR spectra were measured on a Bruker 300 MHz spectrometer
using CD3OD or CDCl3 as solvents. MWs and MW distributions were de-
termined by a GPC equipped with a Waters 1515 pump and a Waters
2414 differential refractive index detector (set at 308C). It used a series
of three linear Styragel columns (HT2, HT4, and HT5) at an oven tem-
Biotin azide was synthesized by the amidation reaction of
biotin with 1-azido-3-aminopropane (Scheme 3a). Previous-
ly, Hsu et al. reported the synthesis of biotin azide by utiliz-
ing o-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hex-
afluorophosphate (HBTU) as the catalyst, but the yield was
only about 40% after purification.[25] We slightly modified
the experimental procedures by activating biotin with CDI
for 3 hours and the yield dramatically increased to 90.7%.
perature of 458C. The eluent was DMF at a flow rate of 1.0 mLminꢀ1
.
FTIR spectra were measured on a Bruker Vector 22 Fourier transform
infrared spectrometer using the KBr disk method. UV/Vis spectra were
recorded on a Unico UV-2102 PC spectrophotometer. Matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry (MALDI-
TOF MS) were recorded in the linear mode on a Bruker BIFLEXe III
using a nitrogen laser (337 nm) and an accelerating potential of 20 kV.
2,5-Dihydroxybenzoic acid (DHB; Aldrich) was used as the matrix and
NaBF4 was added to improve ionization. For diffractive optics technology
(DOT) measurements, dotLab data were recorded on a DOT system
from Axela Biosensors; the sensor chip was composed of polystyrene as
the substrate and avidin groups were attached on the surface. Phosphate
buffered saline (PBS) was used as the eluent. Aqueous solutions of bio-
tinylated polymers were loaded into the sensor chip according to the de-
signed program. The binding events were taking place on the surface of
the sensor chip by avidin–biotin interactions and the height of the diffrac-
tive pattern was increased. The increase in signals was detected synchro-
nously by using a laser-based optical system, and the real-time data gen-
erated were presented in dotLab software.
1
The H NMR spectrum of biotin azide is shown in Figure 1a
and the signal integral ratios confirmed the successful prepa-
ration of biotin azide.
The alkynyl-terminated precursor, alkynyl-PNIPAM, was
synthesized by ATRP of NIPAM monomer by using prop-
argyl 2-chloropropionate and CuCl/Me6TREN as initiator
and catalyst, respectively (Scheme 3b). This polymerization
system is successful and efficient for the NIPAM monomer
and the DP can be facilely adjusted by the monomer/initia-
tor feed monomer ratio and monomer conversion.[22] The
1H NMR spectrum of alkynyl-PNIPAM is shown in Fig-
ure 1b. Resonance signals at d=4.7 ppm (signal a) are as-
cribed to methylene protons of the terminal propargyl
group and signals at d=3.9–4.1 ppm (signal d) are assigned
to the methenyl proton adjacent to amide residues in the
PNIPAM segment. On the basis of the integral ratio of sig-
Results and Discussion
Each avidin molecule contains a maximum of four biotin
binding sites with a high binding constant (Kd ꢁ10ꢀ15 m) and
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Figure 1. 1H NMR spectra recorded for a) biotin azide in [D4]MeOH,
b) alkynyl-PNIPAM19 in CDCl3, and c) biotin-PNIPAM19 in [D4]MeOH.
Figure 2. A) GPC traces in DMF obtained for a) alkynyl-PNIPAM19 and
b) biotin-PNIPAM19. B) FTIR spectra obtained for a) biotin azide, b) al-
kynyl-PNIPAM19, and c) biotin-PNIPAM19.
nals a to d, the DP of alkynyl-PNIPAM was determined to
be 19, thus the polymer was denoted as alkynyl-PNIPAM19.
The GPC trace of the alkynyl-PNIPAM19 precursor in DMF
is shown in Figure 2A, which reveals a relatively sharp and
symmetric elution peak with no tailing or shoulder at lower
or higher MWs, thus indicating that the ATRP process was
conducted in a controlled manner. The MALDI-TOF mass
spectrum of alkynyl-PNIPAM19 reveals an envelope of sig-
nals extending from 1000 to 4000 Da with an Mn,MALDI of
2300 Da and an Mw/Mn of 1.07 (Figure 3a), which is in good
tion profile was again monomodal and quite symmetric,
thereby implying that the click reaction went to completion
with high efficiency. The MALDI-TOF mass spectrum of
biotin-PNIPAM19 presents a series of signals with the high-
est intensity located at m/z 2605.2 (Figure 3b). Compared
with that of the alkynyl-PNIPAM19 precursor, a clear and
systematic shift in the MW distribution was observed. For
biotin-PNIPAM19, MALDI-TOF MS analysis gave an
Mn,MALDI of 2600 Da and an Mw/Mn of 1.09. These results
confirmed the successful preparation of biotin-PNIPAM19.
Moreover, FTIR absorbance peaks characteristic of azide
and alkynyl moieties within biotin azide and alkynyl-
PNIPAM19, respectively, at 2100 cmꢀ1 completely disap-
peared after the click reaction (Figure 2B), suggesting
excess biotin azide was completely removed. By following
similar procedures, biotin-PNIPAM50 with an Mn,GPC of
7400 Da and an Mw/Mn of 1.08 was also synthesized. Struc-
tural parameters are summarized in Table 2.
1
agreement with H NMR spectroscopy results (Figure 1b).
Previously, biotin-terminated PNIPAM was synthesized
by a controlled radical polymerization technique starting
from biotin-based initiators.[14b] In the current work, we em-
ployed the click reaction. Upon reacting with an excess of
biotin azide through the click reaction, the chain end of al-
kynyl-PNIPAM19 was functionalized with biotin and this
process afforded biotin-PNIPAM19 (Scheme 3b). The un-
reacted biotin azide residues were eliminated by treatment
with alkynyl-functionalized resin through click grafting fol-
lowed by simple filtration procedures. The 1H NMR spec-
trum of biotin-PNIPAM19 is shown in Figure 1c. Resonance
signals ascribed to the propargyl group shifted from d=4.7
(signal a, Figure 1b) to 5.2 ppm (signal b, Figure 1c), and a
new resonance signal appeared at d=8.14 ppm, which is
characteristic of the formation of 1,2,3-triazole rings. In ad-
dition, the GPC curve of biotin-PNIPAM19 exhibited a dis-
cernible shift towards higher MW (Figure 2A), and the elu-
For the synthesis of biotin-terminated diblock copolymer,
biotin-PNIPAM-b-PDMA, the alkynyl-terminated precursor
alkynyl-PNIPAM-b-PDMA was synthesized first by ATRP
of the DMA monomer by utilizing alkynyl-PNIPAM19 as the
1
macroinitiator (Scheme 3b). The H NMR spectrum and the
associated signal assignments of alkynyl-PNIPAM-b-PDMA
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Construction of Polymer–Protein Bioconjugates
binding affinity between avidin and biotinylated polymers,
biotin-PNIPAM50 with an Mn,GPC of 7400 Da and Mw/Mn of
1.08, and biotin-PNIPAM69-b-PDMA65 with an Mn,GPC of
13800 Da and Mw/Mn of 1.06 were also synthesized. Their
structural parameters are summarized in Table 2.
Note that in the above-prepared biotin-PNIPAM and
biotin-PNIPAM-b-PDMA, the biotin functionality was lo-
cated at the chain terminal of the PNIPAM homopolymer
and PNIPAM-b-PDMA diblock copolymer. We further pre-
pared PNIPAM-biotin-PNIPAM and PEO-biotin-PNIPAM
in which the biotin moiety was located at the chain middle
of the PNIPAM homopolymer and the diblock junction
point, respectively. For the synthesis of PNIPAM-biotin-
PNIPAM, trifunctional alkynyl-(Cl)2 was prepared first and
utilized as the ATRP initiator for the polymerization of the
NIPAM monomer, thus affording PNIPAM-alkynyl-
PNIPAM (Scheme 3c). The polymerization conditions were
similar to those employed for the synthesis of alkynyl-
PNIPAM, as described in the previous section. The overall
DP of PNIPAM-alkynyl-PNIPAM was calculated to be 54,
according to 1H NMR spectroscopy results (Figure S3a in
the Supporting Information). Thus, the alkynyl-functional-
ized polymer was denoted as PNIPAM27-alkynyl-PNIPAM27.
The subsequent click reaction of PNIPAM27-alkynyl-
PNIPAM27 with biotin azide afforded PNIPAM27-biotin-
PNIPAM27. In the 1H NMR spectrum, a new resonance
signal at dꢁ8.08 ppm could be discerned, which verified the
successful click reaction between biotin azide and alkynyl
moiety (Figure S3b in the Supporting Information). GPC
elution traces of PNIPAM27-alkynyl-PNIPAM27 and
PNIPAM27-biotin-PNIPAM27 also revealed monomodal and
symmetric peaks in both cases and the Mn of the latter shift-
ed slightly from 6200 to 6400 (Figure S4 in the Supporting
Information).
Figure 3. MALDI-TOF mass spectra recorded for a) alkynyl-PNIPAM19
and b) biotin-PNIPAM19.
are shown in Figure S1a in the Supporting Information. On
the basis of signal integral ratios, the DP of the PDMA
block was determined to be eight, thus the diblock copoly-
mer was denoted as alkynyl-PNIPAM19-b-PDMA8. The
GPC trace of alkynyl-PNIPAM19-b-PDMA8 exhibited a
clear shift towards higher MW relative to that of alkynyl-
PNIPAM19, and the peak is reasonably symmetric without
any tailing, indicating a successful chain extension of the
PDMA block (Figure S2 in the Supporting Information).
Subsequently, biotin-PNIPAM19-b-PDMA8 was synthe-
sized by the click reaction of biotin azide with alkynyl-
PNIPAM19-b-PDMA8 by following similar procedures as
those employed for the preparation of biotin-PNIPAM19.
Resonance signals ascribed to biotin azide can be clearly
seen in the NMR spectrum of alkynyl-PNIPAM19-b-PDMA8
(Figure S1b in the Supporting Information). Compared with
the alkynyl-PNIPAM19-b-PDMA8 precursor, a new reso-
nance signal appeared at d=8.10 ppm, which was character-
istic of the formation of 1,2,3-triazole linkages; this again
implied the successful synthesis of biotin-PNIPAM19-b-
PDMA8. Compared with that of alkynyl-PNIPAM19-b-
PDMA8, the GPC trace of biotin-PNIPAM19-b-PDMA8 in
DMF exhibited a slight shift towards higher MW (Figure S2
in the Supporting Information). In addition, the GPC trace
was also monomodal and quite symmetric, suggesting that
the click reaction did not affect the structural integrity. For
comparison, and the investigation into MW effects on the
Finally, PEO-biotin-PNIPAM was prepared by the click
reaction of PEO-alkynyl-PNIPAM with biotin azide
(Scheme 3d). 3,5-Bis(propargyloxy)benzyl alcohol was pre-
pared according to literature procedures.[20] Diblock copoly-
mer with one single alkynyl moiety at the junction point,
PEO-alkynyl-PNIPAM, was then synthesized in three steps.
First, PEO-alkynyl-OH was synthesized by the click cou-
pling reaction between PEO-N3 and an excess of 3,5-bis(-
propargyloxy)benzyl alcohol (20 equiv) under dilute condi-
tions to assure that only one alkynyl group participated in
1
the click reaction. The H NMR spectrum of PEO-alkynyl-
OH is shown in Figure 4a, together with signal assignments.
Resonance signals characteristic of PEO and 3,5-bis(propar-
gyloxy)benzyl alcohol can be clearly observed and the inte-
gral ratio of signal f to signal g is about 2:1; this confirmed
the successful synthesis of PEO-alkynyl-OH. Compared
with that of the PEO-N3 precursor, the GPC trace of PEO-
alkynyl-OH in DMF exhibited a slight shift towards higher
with no tailing or shoulders (Figure 5A). This result further
indicated that only one alkynyl group in 3,5-bis(propargy-
loxy)benzyl alcohol was consumed during the click reaction;
otherwise, we would discern a shoulder peak corresponding
to the coupled product, namely, PEO-OH-PEO.
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The hydroxyl functionality of PEO-alkynyl-OH was then
treated with 2-chloropropionyl chloride to afford the ATRP
macroinitiator, PEO-alkynyl-Cl. After the esterification re-
action, the resonance signal of l shifted from d=4.69 to
5.19 ppm (Figure 4b), and a new resonance signal ascribed
to methyl protons (signal r) appeared at dꢁ1.8 ppm. The in-
tegral ratio of signal j to signal r was about 2:3, suggesting
completion of the esterification reaction. The GPC elution
trace of PEO-alkynyl-Cl revealed a narrow and symmetric
peak with no tailing or shoulder at lower or higher MWs
(Figure 5A).
Subsequently, PEO-alkynyl-PNIPAM was obtained by
ATRP of the NIPAM monomer by using PEO-alkynyl-Cl as
the macroinitiator. On the basis of the integral ratio of
signal f, which was characteristic of PNIPAM, to signal a,
which was characteristic of PEO sequences, the DP values
of the PNIPAM block were estimated to be 45 (Figure 6a).
Thus, the product was denoted as PEO45-alkynyl-PNIPAM45
(Table 1). Compared with the PEO-alkynyl-Cl precursor,
the GPC trace of PEO-alkynyl-PNIPAM in DMF clearly
shifted to higher MW, yielding Mw/Mn of 1.10 (Figure 5A).
In the final step, PEO-alkynyl-PNIPAM was biotinylated by
Figure 4. 1H NMR spectra recorded in CDCl3 for a) PEO45-alkynyl-OH
and b) PEO45-alkynyl-Cl.
1
a click reaction with biotin azide. The H NMR spectrum of
PEO-biotin-PNIPAM is shown in Figure 6b. Compared with
that of PEO-alkynyl-PNIPAM, the resonance signal ascribed
to alkynyl moieties (signal c) almost disappeared and the
signal ascribed to the proton in 1,3-triazole rings was consid-
erably enhanced; this indicated the successful preparation of
PEO-biotin-PNIPAM. Moreover, the GPC trace of PEO-
biotin-PNIPAM in DMF exhibited a slight shift to higher
MW associated with a narrow polydispersity of 1.10 (Fig-
ure 5A). The efficiency of the click reaction was further
confirmed by FTIR analysis (Figure 5B), as evidenced by
the complete disappearance of characteristic azide alkynyl
moieties at about 2100 cmꢀ1).
Figure 5. A) GPC traces recorded in DMF for a)PEO45-N3, b)PEO45-al-
kynyl-OH,
c)PEO45-alkynyl-Cl,
d)PEO45-alkynyl-PNIPAM45,
and
e)PEO45-biotin-PNIPAM45. B) FTIR spectra recorded for a)biotin azide,
b)PEO45-alkynyl-PNIPAM45, and c)PEO45-biotin-PNIPAM45.
Figure 6. 1H NMR spectra recorded in CDCl3 for a) PEO45-alkynyl-
PNIPAM45 and b) PEO45-biotin-PNIPAM45.
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Construction of Polymer–Protein Bioconjugates
Effects of Polymer MWs and Steric Hindrance on the
Binding Numbers of Hybrid Polymer/Avidin Bioconjugates
Avidin is a glycoprotein consisting of four identical subunits
and each subunit can strongly bind to one biotin with high
affinity.[6] By taking advantage of this special interaction be-
tween avidin and biotin, we can facilely fabricate star poly-
mers, star block copolymers, and heteroarm star copolymers
starting from biotinylated polymers with the biotin function-
ality located at the chain terminal and chain middle of ho-
mopolymers and diblock copolymers (Table 2 and
Scheme 2). A closer examination of the seven samples re-
vealed that they could serve as a series of excellent systems
for the investigation of polymer MW effects and steric hin-
drance effects on the binding affinity between avidin and
biotinylated polymers. The binding numbers were then
quantified by standard HABA/avidin assays.
The HABA/avidin reagent is quite sensitive and conven-
ient for the spectroscopic examination of avidin/biotin bind-
ing events. In the absence of biotin or biotinylated polymers,
HABA will specifically form a noncovalent molecular recog-
nition complex with avidin with an association constant (Ka)
of 7ꢁ106 mꢀ1, and the complex exhibits a characteristic opti-
cal adsorption peak at about 500 nm. The addition of biotin
or biotin derivatives to the HABA/avidin assay mixture will
result in the displacement of HABA from the binding sites
because the biotin/avidin Ka value is as high as 1015 mꢀ1. This
displacement event is also accompanied by a proportional
decrease in the optical absorbance at 500 nm. Thus, the
binding number of biotinylated polymer chains per avidin
molecule can be facilely determined.
Figure 7. Variation of optical absorbance of HABA/avidin assay mixture
upon titration with aqueous solutions of a) biotin-PNIPAM19 and
b) PEO45-biotin-PNIPAM45.
During the HABA/avidin assay, aqueous solutions of bio-
tinylated polymers at predetermined concentrations were
added dropwise to the HABA/avidin assay mixture
(900 mL). The variation of optical absorbance at 500 nm was
then recorded as a function of the amount of biotin-
PNIPAM19 aqueous solution added (Figure 7a). Upon addi-
tion of an aqueous solution of biotin-PNIPAM19 (10–
300 mL), the absorbance at 500 nm decreased almost linear-
ly, indicating that biotin-PNIPAM19 continuously binds
avidin and replaces HABA from the initially formed
HABA/avidin complex. Upon further addition of biotin-
PNIPAM19, the decrease of optical absorbance slowed down
considerably, indicating that the binding of more biotin-
PNIPAM19 chains onto avidin was getting difficult. At later
stages, the decrease could be mainly ascribed to the dilution
effect. From the intersection point of the two lines, showing
the changing trends at early and later stages of biotin-
PNIPAM19 addition (Figure 7a), the amount of biotin-
PNIPAM19 needed to maximally replace HABA molecules
from the initially formed HABA/avidin complex can then
be calculated. The binding number of biotin-PNIPAM19 per
avidin was thus determined to be about 3.3 (Table 2).
binding number is four. Another typical example is shown
for PEO45-biotin-PNIPAM45, in which the biotin functionali-
ty is located at the junction point. From Figure 7b, we can
tell that the trend of changes in optical absorbance with
gradual addition of PEO45-biotin-PNIPAM45 is quite similar
to that exhibited by biotin-PNIPAM19. Apparently, the tran-
sition from quick decrease to slow decrease in optical ab-
sorbance occurs at much lower volume of PEO45-biotin-
PNIPAM45 solution added than that for biotin-PNIPAM19.
Accordingly, the binding number of PEO45-biotin-
PNIPAM45 chains per avidin was determined to be 2.2
(Table 2) and this clearly implied the steric hindrance effect.
When the biotin moiety was located at the diblock junction,
its binding with avidin will be opposed by the closely con-
nected PEO and PNIPAM blocks. The above results partial-
ly confirmed our initial consideration. Also, note that based
on the above description, heteroarm star copolymers have
about 2.2 PEO and about 2.2 PNIPAM arms formed by
binding between PEO45-biotin-PNIPAM45 and avidin.
Subsequently, the binding numbers for all biotinylated
polymers during their conjugation with avidin were deter-
mined and the results are summarized in Table 2. For biotin-
PNIPAM19, biotin-PNIPAM50, and biotin-PNIPAM69-b-
PDMA65, the average binding numbers decreased from 3.3
to 2.9 to 1.9, respectively. This clearly suggested that the
The above results indicated that the steric hindrance ex-
erted by the covalently attached PNIPAM19 chain almost
did not inhibit the affinity binding between biotin-
PNIPAM19 and avidin, although the theoretical maximum
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2843

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higher polymer MWs, the lower binding affinity between
avidin and biotin-terminated PNIPAM homopolymer or
PNIPAM-b-PDMA diblock copolymer. A further compari-
son between the binding numbers of biotin-PNIPAM50 and
PNIPAM27-biotin-PNIPAM27, 2.9 and 1.9, respectively, re-
vealed that the placement of the biotin functionality at the
chain middle could dramatically decrease its binding tenden-
cy towards avidin because the two polymers possess similar
chain lengths and only differ in the location of the biotin
functionality within the chain. For PEO45-biotin-PNIPAM15
and PEO45-biotin-PNIPAM45, the biotin functionalities are
both located at the diblock junction and the corresponding
binding numbers are 2.5 and 2.2, respectively; this again re-
flected the effect of polymer MW of the biotinylated precur-
sors on the binding process.
Figure 8. DotLab responses at the surface of an avidin-coated sensor chip
upon injection of aqueous solutions of biotin-PNIPAM19 and PNIPAM27-
biotin-PNIPAM27.
qualitative agreement with the difference in the correspond-
ing binding numbers (Table 2).
Moreover, the binding numbers of biotin-PNIPAM50 and
biotin-PNIPAM19-b-PDMA8 per avidin are both 2.9, which
might be a coincidence and partially imply that PDMA
chain conformations, noting that DMA is an N,N-dialkyl-
substituted monomer, provide more steric hindrance to the
avidin/biotin binding process than that exerted by the
PNIPAM sequence. Another interesting comparison be-
tween PNIPAM27-biotin-PNIPAM27 (binding number 1.9)
and PEO45-biotin-PNIPAM15 (binding number 2.5) further
revealed that the PEO sequence exerted much lower steric
hindrance to the molecular recognition event than that ex-
erted by PNIPAM; this may be because the PEO chain con-
formation is quite flexible and contains no side groups.[17]
We can thus deduce that for biotinylated PEO, PNIPAM,
and PDMA of comparable chain lengths, the binding affinity
with avidin decreases in the order of PEO>PNIPAM>
PDMA owing to the steric hindrance effect of varying chain
conformations and the absence or presence of side groups.
Overall, the above results successfully interpreted the steric
hindrance effects of polymer MWs, biotin location within
the chain, and chain conformations of biotinylated polymers
on their binding affinities, that is, binding number, with
avidin. Note that the determination of binding numbers can
also tell us the exact number of noncovalently grafted poly-
mer arms per avidin within the supramolecular star poly-
mers, star block copolymers, and heteroarm star copolymers
(Schemes 1 and 2).
Finally, the availability of biotin moieties on biotinylated
polymers for specific conjugation to surface-bound avidin
and the binding process were further examined by using a
diffractive optics technology (dotLab) system (Figure 8);
this is a sensitive technique to monitor small changes in dif-
fraction owing to binding events occurring at the surface of
a sensor chip.[6e,26] As shown in Figure 8, a clear response is
observed after the injection of aqueous solutions of either
biotin-PNIPAM19 or PNIPAM27-biotin-PNIPAM27. Most im-
portantly, the signal increase retained to a large degree after
washing. Qualitatively, we can also tell from Figure 8 that
PNIPAM27-biotin-PNIPAM27 apparently exhibits a smaller
response upon injection than that of biotin-PNIPAM19,
which suggested that the former exerts a larger steric hin-
drance effect during conjugation with avidin, and this is in
Conclusion
A series of homopolymers and block copolymers site-specif-
ically labeled with one single biotin functionality at the
chain terminal, chain middle, or diblock junction point were
synthesized by the combination of ATRP and click chemis-
try. By taking advantage of the specific noncovalent interac-
tion between avidin and biotin moieties, polymer–protein
bioconjugates with varying star-type topologies, including
star polymers, star block copolymers, and heteroarm star
polymers, were fabricated. The steric hindrance effects
(polymer MW, location of biotin moiety within the chain,
and polymer chain conformations) on the conjugation effi-
ciency between biotinylated polymers and avidin were sys-
tematically explored. Standard avidin/HABA assays re-
vealed that polymer MW and the location of biotin moiety
within the polymer chain played crucial roles in the binding
affinity, and the binding numbers varied from 3.3 to 1.9. For
biotinylated polymers with similar chain architectures, but
different MWs, those with larger MWs exhibited a lower
binding affinity with avidin. For biotinylated polymers with
comparable MWs, but different chain architecture, those
with the biotin functionality located at the chain middle or
diblock junction point possessed higher steric hindrance
during conjugation with avidin than those of biotin-termi-
nated polymers. Finally, for biotinylated PEO, PNIPAM,
and PDMA of comparable chain lengths, the binding affinity
with avidin decreased in the order of PEO>PNIPAM>
PDMA through the steric hindrance effect as a result of dif-
ferent chain conformations and the absence or presence of
side groups.
Acknowledgements
The financial support from the National Natural Scientific Foundation of
China (NNSFC) Project (20874092, 91027026, and 51033005) and the
Fundamental Research Funds for the Central Universities is gratefully
acknowledged.
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Construction of Polymer–Protein Bioconjugates
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Received: May 27, 2011
Published online: September 2, 2011
Chem. Asian J. 2011, 6, 2835 – 2845
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
2845