Biotin functionalized poly(sulfonic acid)s for bioconjugation: In situ binding monitoring by QCM-D

Article abstract of DOI:10.1002/pola.24532

We describe the synthesis of biotin end functionalized poly(sulfonic acid)s via living radical polymerization (LRP) for conjugation to Avidin. Quartz crystal microbalance (QCM-D) and competitive binding studies were used to confirm this conjugation. A biotin initiator for copper-mediated LRP was used to provide acrylamide and methacrylate based polymers with the functional end group. This investigation revealed that 2-acrylamido-2-methyl-1-propanesulfonic acid was not a suitable monomer in its acid form but was successfully used in its sodium salt form. A second monomer, 3-sulfopropylmethacrylate as the potassium salt was also studied and both monomers produced polymers with polydispersities <1.3 and 1.4, respectively. Evolution of molecular weight with respect to time indicated that the polymerization of the acrylamide polymer is controlled. Quartz crystal microbalance with dissipation monitoring was used to confirm that the biotinylated polymers were able to bind to Avidin in situ. The gold surface of a quartz crystal was chemically modified resulting in a stable monolayer of Avidin; the biotinylated polymers were passed over the functionalized surface and their grafting ability was examined. A competitive binding evaluation was undertaken with 2-(4-hydroxyphenylazo)benzoic acid (HABA) dye to provide visual verification of conjugation.

Full text of DOI:10.1002/pola.24532

Biotin Functionalized Poly(sulfonic acid)s for Bioconjugation: In Situ
Binding Monitoring by QCM-D
STACY SLAVIN,1 ANNE DE CUENDIAS,1 V. LADMIRAL,2 DAVID M. HADDLETON1
1Department of Chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom
2Department of Chemistry, Dainton Building, University of Sheffield, Brook Hill, Sheffield, S3 7HF, United Kingdom
Received 2 November 2010; accepted 24 November 2010
DOI: 10.1002/pola.24532
Published online 3 January 2011 in Wiley Online Library (wileyonlinelibrary.com).
ABSTRACT: We describe the synthesis of biotin end functional-
ized poly(sulfonic acid)s via living radical polymerization (LRP)
for conjugation to Avidin. Quartz crystal microbalance (QCM-D)
and competitive binding studies were used to confirm this con-
jugation. A biotin initiator for copper-mediated LRP was used
to provide acrylamide and methacrylate based polymers with
the functional end group. This investigation revealed that 2-
acrylamido-2-methyl-1-propanesulfonic acid was not a suitable
monomer in its acid form but was successfully used in its so-
dium salt form. A second monomer, 3-sulfopropylmethacrylate
as the potassium salt was also studied and both monomers
produced polymers with polydispersities <1.3 and 1.4, respec-
tively. Evolution of molecular weight with respect to time indi-
cated that the polymerization of the acrylamide polymer is
controlled. Quartz crystal microbalance with dissipation moni-
toring was used to confirm that the biotinylated polymers were
able to bind to Avidin in situ. The gold surface of a quartz crys-
tal was chemically modified resulting in a stable monolayer of
Avidin; the biotinylated polymers were passed over the func-
tionalized surface and their grafting ability was examined. A
competitive binding evaluation was undertaken with 2-(4-
hydroxyphenylazo)benzoic acid (HABA) dye to provide visual
C
V
verification of conjugation.
2011 Wiley Periodicals, Inc.
J Polym Sci Part A: Polym Chem 49: 1163–1173, 2011
KEYWORDS: biotin–avidin; heteroatom-containing polymers;
living polymerization; water-soluble polymers
INTRODUCTION Molecular recognition is of major impor-
tance in that it plays a part in many biological interactions.
The avidin–biotin interaction is an excellent model for study-
ing biorecognition due to the ultra-high binding affinity
between them. Avidin is a homotetrameric protein composed
of four identical sub-units, each of these units can bind with
one biotin molecule; this interaction is considered to be one
of the strongest non-covalent biological interactions known.1
Bioconjugation of functional polymers to proteins results in
bioconjugates with new properties, which can provide poten-
tial benefits. A number of reviews discuss the advantages of
increased biological activity and stability; both of importance
for therapeutic applications.2–11 The biotin–avidin interaction
is of high interest in biological applications and hence the
ability to incorporate biotin into polymers is of use.
llough Grignard metathesis reaction and chemical vapor
deposition polymerization using Huisgen click chemistry for
the modification has been reported.17–19
LRP of methacrylate and acrylamide based monomers con-
taining sulfonic acid salts are reported in this article with
biotin incorporated into the initiator. The use of copper(0)
as a source of copper as opposed to copper(I) salts has
emerged as en effective system for the polymerization of
vinyl monomers in polar solvents such as water.20,21 Sulfonic
acids are of interest since they have been shown to form
excellent hydrophilic polymers with no toxicity and to inter-
act with fibroblast growth factor (FGF), thus inhibiting the
intracellular signaling.22,23 In terms of monomer design, ac-
rylamide monomers are useful in a biological sense due to
their thermoresponsive properties however it is well
reported in literature that their polymerization via ATRP can
be challenging.24 Sulfonic acid containing monomers are gen-
erally polymerized by free radical polymerizations.22,23,25–28
In addition, sulfonic acids can be introduced via post poly-
merization modification.29,30 The quartz crystal microbalance
(QCM) technique measures a mass per unit area by meas-
uring the change in the quartz crystal resonator. The reso-
nance is disturbed by the addition or removal of a small
mass due to oxide growth/decay or film deposition at the
Biotin has been incorporated into polymers in a number of
ways. Polymers made via atom transfer radical polymeriza-
tion, LRP, have incorporated biotin by both an initiator and
via post polymerization modification.12,13 Biotinylated
polymers have also been synthesized via reversible addition-
fragmentation chain transfer, RAFT, with
a biotinylated
RAFT chain transfer agent or post polymerization modifica-
tion.14–16 Post polymerization modification with commer-
cially available polyester (PLGA) polymers, from the McCu-
Correspondence to: D. M. Haddleton (E-mail: d.m.haddleton@warwick.ac.uk)
C
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Journal of Polymer Science Part A: Polymer Chemistry, Vol. 49, 1163–1173 (2011) 2011 Wiley Periodicals, Inc.
BIOTIN FUNCTIONALIZED POLY(SULFONIC ACID)S FOR BIOCONJUGATION, SLAVIN ET AL.
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JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
SCHEME 1 Synthesis of biotin initiator: (i) Kundiger method32: H2SO4, from 0 ꢀC to 25 ꢀC; (ii) SOCl2, from 0 ꢀC to 25 ꢀC; (iii) TEA,
THF, from 0 ꢀC to 20 ꢀC; (iv) Yamaguchi procedure: TEA, DMF, from 0 ꢀC to 20 ꢀC. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
surface of the acoustic resonator. The dissipation quantifies
the damping in the system, and gives information on the
sample’s viscoelastic properties.
Polymer Synthesis
a-Biotin Poly(3-sulfopropylmethcrylate,
potassium), a-Biotin pSPMA
a-Biotin pSPMA (5), Scheme 2, was synthesized via LRP
using 3-sulfopropylmethacrylate, potassium. Polymerization
was carried out in a H2O/MeOH/DMSO solvent mixture at
RESULTS AND DISCUSSION
Previous studies have demonstrated that biotinylated den-
drons can be synthesized for bioconjugation to avidin.31 Bio-
tin was modified into a non-symmetrical initiator, which was
transformed via a Michael addition with methyl acrylate and
amidation with an excess of ethylenediamine to double the
number of surface amine groups. This was repeated until the
required dendron size was reached. This present study
reports the modification of biotin to an initiator suitable for
living radical polymerization (LRP) to polymerize sulfonic
acid monomers to polymers with a comb-like architecture.
After synthesis, the polymers biological activity was investi-
gated using quartz crystal microbalance with dissipation
monitoring, QCM-D.
ꢀ
40 C to ensure complete solubilization of all reagents. After
19 h, a conversion of 77% was reached and the polymeriza-
tion was stopped. Purification by precipitation and dialysis
led to a polymer with Mn ¼ 22,000 g/mol (Table 1).
a-Biotin Poly(2-acrylamido-2-methyl-1-propanesulfonic
acid, sodium), a-Biotin pAAMPSA
Following on from this synthesis, acrylamide monomers
were investigated; some containing protected sulfonic
Initiator Synthesis
The polymerization initiator was synthesized via modifica-
tion of the valeric acid side chain of biotin via a Yamaguchi
esterification reaction, Scheme 1. Standard DCC coupling pro-
vided low yielding products and EDCI coupling gave higher
yields but also resulted in halogen exchange taking place.
The esterification method has been used by Wullschleger
et al. for the synthesis of an E-configured macrolactone
cyclization product whereas Yamaguchi et al. developed the
procedure for the synthesis of large-ring lactones.33,34 The
reaction proceeds via the formation of biotin-2,4,6-trichloro-
benzoic mixed anhydride, followed by the nucleophilic attack
of the alcohol, bearing the initiator function, to the biotin
carboxylic group. Maynard et al. have shown the synthesis of
biotin initiators via an amide linkage for increased hydrolytic
stability; however, in this report, the stability of the ester
link was sufficient over the duration of the reactions.13,15
SCHEME 2 (i) Polymerization of a-biotin poly(3-sulfopropyl-
methcrylate, potassium) by LRP: N-(n-ethyl)-2-pyridylmethani-
mine, CuBr, CuBr2, H2O/MeOH/DMSO, 40 ꢀC. [Color figure can
be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
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TABLE 1 Data for the Polymers Synthesized
a
b
Biotin
Mn(theory)
Mn(NMR)
Mn(GPC)
Polymer
Conv. (%)
DP
(g/mol)
(g/mol)
(g/mol)
MW/Mn
5: pSPMA
77
75
72
90
77
74
25,100
23,400
23,400
22,600
18,000
17,500
20,700
57,800
36,600
1.4
2.3
1.3
13A: pAAMPSA
13B: pAAMPSA
a
At 100% conversion.
b
Characterization of polymers by 1H NMR (D2O) and by GPC (NaNO3/NaH2PO4 in water, PEG calibration).
groups, Scheme 3, with the sodium salts giving the best con-
plot (a) Figure 1 is relatively linear however, the evolution of
Mn with time (b) is typical of a conventional free radical po-
lymerization as opposed to a controlled/living polymeriza-
tion and we were not able to determine if the Cl remained
on the polymer terminus.
trol with standard initiators. Polymerizations of N,N-dimethy-
ꢀ
lacrylamide were studied in DMSO at 30 C, using Me6-TREN
and CuCl(ii, Scheme 3).35
The polymerizations in i, Scheme 3, reveal that the sulfonic
acid monomer (6) is difficult to polymerize however, fast
polymerization was achieved when using the commercially
available monomer which is supplied in 50 wt % aqueous
solution (11); once lyophilized to use the monomer in its
powder form. The first investigation of the salt monomer, iii,
Scheme 3 does not follow first order kinetics but formed an
ill defined polymer in DMSO/water solution.
After 7 h, a conversion of 75% was reached and the poly-
merization was stopped. Purification by precipitation and di-
alysis led to a polymer with Mn ¼18,000 g/mol (Table 1)
with a PDI ¼ 2.3, thus this route is not an ideal way to
obtain a controlled polymerization of this monomer.
Route B was finally undertaken, repeating polymerization iii
(Scheme 3) but without the presence of water. In this case,
curvature can be seen in the kinetic plot Figure 2 this can be
attributed to catalyst deactivation or termination events.13,35
After 5 h and 20 min of polymerization, a conversion of
72% was reached and the polymerization was stopped. Puri-
fication by precipitation and dialysis led to a polymer with a
Mn ¼ 18,000 g/mol (Table 1). A fairly narrow PDI of 1.3 was
Route A (Scheme 4) by LRP was also investigated. Inꢀ this
case, the polymerization of (11) was carried out at 30 C in
a solvent mixture of MeOH/DMSO/H2O with CuCl/N-(n-
ethyl)-2-pyridylmethanimine as the catalyst. This polymeriza-
tion was slower and allowed for sampling during the reac-
tion to investigate the kinetics of the reaction. The kinetic
SCHEME 3 Polymerization tests: (i) Polymerization of 2-acrylamido-2-methyl-1-propanesulfonic acid by LRP: Me6-TREN, CuCl,
DMSO, 30 ꢀC; (ii) N,N-dimethylacrylamide by LRP: Me6-TREN, CuCl, DMSO, 30 ꢀC; (iii) 2-acrylamido-2-methyl-1-propanesulfonic
acid, sodium salt by LRP: Me6-TREN, CuCl, H2O/DMSO, 30 ꢀC. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.].
BIOTIN FUNCTIONALIZED POLY(SULFONIC ACID)S FOR BIOCONJUGATION, SLAVIN ET AL.
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JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
Quartz Crystal Microbalance with Dissipation Monitoring
QCM with dissipation monitoring is a technique developed
by Kasemo et al. used to study molecular interactions and
molecular adsorption at surfaces.36–38 It determines the
mass of very thin surface bound layers and simultaneously
gives information about their structural and viscoelastic
properties. It is a useful technique as the sensor mimics
real-life applications, giving real-time interface characteriza-
tion and evaluation of kinetics.39
The gold surface of the QCM sensors was chemically modi-
fied to provide a firmly bound avidin surface Scheme 5.40 Af-
ter which frequency and dissipation measurements relating
to the interaction between the avidin and biotinylated poly-
mers were collected. When Avidin was applied via adsorp-
tion, the binding was not as strong as the Avidin reaction
with the biotinylated polymer. This resulted in the polymer
‘‘stripping’’ the Avidin from the surface which could be seen
from an unusual increase in frequency.
SCHEME 4 Polymerization of 2-acrylamido-2-methyl-1-propane-
sulfonic acid, sodium salt, Route A by LRP N-(n-ethyl)-2-pyridyl-
methanimine, CuCl, H2O/MeOH/DMSO, 30 ꢀC; Route B by LRP:
Me6-TREN, CuCl, DMSO, 25 ꢀC. [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
Avidin in PBS buffer, pH 7, was passed over the QCM crystal
in situ to ensure maximum surface coverage of Avidin and a
change in frequency (Fig. 3) showed surface adsorption onto
the crystal surface and a steady state was reached indicated
after ꢁ500 s. Following a buffer rinse, slight increase in fre-
quency corresponding to the removal of unbound Avidin a
steady state was reached after ꢁ2000 s. On addition of a-Bi-
otin pSPMA, a sharp decrease in frequency was observed.
The rate of the Avidin-a-Biotin pSPMA binding is related to
the gradient until a plateau is reached, Figure 3. No polymer
was removed from the surface with a buffer rinse cycle,
showing that the biotinylated polymer is firmly bound to the
Avidin surface.
obtained showing that this procedure is a better route for
the polymerization of this acrylamide.
Although the large molecular weight polymers complicate
the 1H NMR spectra analysis of the small biotin end-group
signals, the theoretical Mn values (calculated at 100%) are
reasonably close in value to the actual Mn values showing
the initiator is fairly efficient (Table 1). For polymer 5, Mn
results from NMR and GPC are close; however, this is not the
case for polymers 13A and 13B. This is due to the different
nature of the poly(sulfonic acid) and its self-organization in
water which leads to higher Mn values in aqueous GPC. An
added effect on the Mn values by GPC is caused by the differ-
ence in structure between the sulfonic acid polymers and
the PEG polymers used to calibrate the aqueous GPC system.
The same sequence was carried out with a-Biotin pAAMPSA
(13b) and a sharp decrease in frequency was observed as
with a-Biotin pSPMA. A very steep gradient is seen as a-
FIGURE 1 Kinetic plot of pAAMPSA (a) and evolution of Mn and polydispersity index (PDI) with time (b). Synthesis conditions:
Route A by LRP: N-(n-ethyl)-2-pyridylmethanimine, CuCl, H2O/MeOH/DMSO, 30 ꢀC. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
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ARTICLE
FIGURE 2 Kinetic plot of pAAMPSA (a) and evolution of Mn and PDI with conversion (b). Synthesis conditions: Route B by LRP:
Me6-TREN, CuCl, DMSO, 25 ꢀC. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Biotin pAAMPSA is adsorbed on the Avidin surface (Fig. 4)
indicating a very fast rate of reaction. No change in fre-
quency was observed during the final buffer rinse indicating
that this polymer is also strongly bound to the Avidin
surface.
of rigid films (which have a negligible dissipation trace)
involves the Sauerbrey relation, which correlates the fre-
quency of the oscillating crystal with the mass deposited on
it. However, if the film is viscoelastic and the polymer film
interacts with the solution being flown over it then the mass
calculated is underestimated and a model must be applied
instead.39,41,42 The data was subjected to both methods to
investigate the mass of biotinylated polymer on the avidin
functionalized surface and give an estimated surface mass
(Table 2; Fig. 5).
A comparison of the two polymers shows that the binding
rate to the Avidin surface is almost identical. The overall
change in frequency following deposition of a-Biotin pSPMA
and a-Biotin pAAMPSA ¼ 8 Hz and 10 Hz, respectively. This
small difference of 2 Hz indicates similar amounts of mass
adsorbed on the surface; however, this is dependent on the
molecular weight as well as the structure. Mass calculation
The Avidin-biotinylated polymer interaction was investigated
under acidic conditions. Initially, PBS buffer pH 7 was passed
SCHEME 5 Gold surface functionalization. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
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JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
FIGURE 4 QCM-D of a-Biotin pAAMPSA (13b). [Color figure can
be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
FIGURE 3 QCM-D Plot for a-Biotin pSPMA (5). [Color figure can
be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
ditions. After the final buffer rinse at pH 4 all of the Avidin
remaining on the surface was completely removed. Thus,
Avidin does not stay bound to the surface at pH 4.
over the Avidin pre-treated crystal and on changing to ace-
tate buffer at pH 4 an increase in frequency was observed,
however, a steady state was not reached. This increase is
ascribed to the acidic conditions removing Avidin from the
surface. A similar trend has been seen previously with Con-
canavalin A, where on lowering the pH of the solution
caused a removal of the protein from a polystyrene coated
quartz crystal.43
HABA Analysis
A competitive binding evaluation was undertaken as an addi-
tional method to confirm the binding ability of the biotinyl-
ated polymers to the protein receptor (Scheme 6). 2-(4-
Hydroxyphenylazo)benzoic acid (HABA) binds to Avidin in a
similar way to biotin and has a maximum UV absorbance at
350 nm. Once complexed to Avidin, the UV absorbance shifts
to 500 nm and a red/orange color is observed in solution.
Avidin’s affinity for HABA is much weaker (Kd ¼ 10ꢂ6 M) in
comparison to biotin (Kd ¼ 10ꢂ15 M) hence HABA should be
replaced by the biotinylated polymers when they are added
to the Avidin-HABA complex.
A solution of Avidin in acetate buffer, pH 4, was passed over
the QCM crystal to ensure maximum surface coverage of Avi-
din. A change in frequency was observed showing mass was
adsorbed on the surface of the crystal and a steady state
was reached indicated by the plateau around 2000 s, Figure
6. After a buffer rinse at pH 4, the frequency increased
showing Avidin was not bound. A steady state was not
reached again nevertheless the overall frequency change at
3500 s was still lower than the initial stage around (1500 s)
before Avidin was passed over the surface. An initial
decrease in frequency indicated adsorption of the polymer
onto the surface however the frequency increase confirmed
that the Avidin is not bound to the surface under these con-
In both cases (a-Biotin pSPMA and a-Biotin pAAMPSA), when
the biotinylated polymer was added to the Avidin-HABA
complex solution, the red/orange color disappeared along
with the peak belonging to the Avidin-HABA complex at k ¼
500 nm and a new peak appeared at k ¼ 350 nm correlating
TABLE 2 Mass Data
Sauerbrey’s
Relation
Model
Polymer Mass
on Surface
(ng/cm2)
x [Excess Avidin
x [Polymer þ Avidin
Mass on
Surface] (ng/cm2)
x [Polymer Mass
on Surface]
(ng/cm2)
Mass on
Surface] (ng/cm2)
Polymer
pH
a-Biotin pSPMA(5)
7
7
230
418
118.54 6 1.65
2296.50 6 44.80
2338.73 6 39.77
2180
985
a-Biotin pAAMPSA (13b)
1353.18 6 14.01
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FIGURE 5 Estimated film mass over time using Sauerbrey’s
relation. [Color figure can be viewed in the online issue, which
is available at wileyonlinelibrary.com.]
FIGURE 6 QCM-D analysis of a-biotin pSPMA (5) at pH 4. [Color
figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
to native HABA, indicating that the biotinylated polymers
have complexed with Avidin (Figs. 7 and 8).
MeOH (3/1.5/0.5 v/v/v) as solvent. The tube was sealed
with a rubber septum and N-(n-ethyl)-2-pyridylmethanimine
ligand (0.023 mL, 0.16 mmol) was injected via a syringe.
The Schlenk solution was subjected to five freeze-pump-
thaw cycles. This solution was then cannulated under nitro-
gen into a second Schlenk tube, previously evacuated and
filled with nitrogen, containing Cu(I)Br (0.0058 g, 0.0406
mmol) and Cu(II)Br2 (0.009 g, 0.0406 mmol). The tempera-
ture was adjusted to 40 ꢀC with constant stirring. Samples
were removed periodically using a degassed syringe for con-
version (t ¼ 0). At the end of the polymerization, the mix-
ture was bubbled with air for 2 h. The polymer was subse-
quently precipitated twice into methanol, dissolved in
deionized water and dialyzed (Spectra/Por 6 standard, re-
generative cellulose membrane, MWCO 8 kDa) for 3 days
and finally freeze-dried. 1H NMR (D2O): The conversion was
Experimental
Reagents
Biotin (Sigma-Aldrich, 99.0%), Avidin (Sigma-Aldrich, 98%),
and 3-sulfopropylmethacrylate, potassium (Sigma-Aldrich,
98%) were used as received, 2-Acrylamido-2-methyl-1-pro-
panesulfonic acid sodium salt solution (Sigma-Aldrich, 50 wt
% in H2O) was lyophilized prior to use and all other
reagents and solvents were obtained at the highest purity
available from Aldrich chemical company and used without
further purification unless stated.
Synthesis of a-Biotin Poly(3-sulfopropylmethacrylate,
potassium) (pSPMA)
SPMA (2 g, 8.1 mmol), biotin initiator (0.039 g, 0.0812
mmol), were charged in a Schlenk tube with H2O/DMSO/
SCHEME 6 HABA schematic.
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JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
FIGURE 7 UV absorbance spectra of HABA analysis of a-Biotin
pSPMA. [Color figure can be viewed in the online issue, which
is available at wileyonlinelibrary.com.]
FIGURE 9 1H NMR spectrum of pSPMA in D2O.
calculated
by
comparing
the
integral
of
the
OACH2ACH2ACH2ASO3K group signals shifting during poly-
merization from 4.31 to 4.17 ppm. Conversion ¼ 77% after
19 h of polymerization, DPn ¼ 90, Mn ¼ 22,600 g/mol. Mn
(GPC, 0.2 M NaNO3, 0.01 M NaH2PO4, PEG calibration) ¼
20,700 g/mol, Mw/Mn (GPC) ¼ 1.4.
OC(O)C(CH3)2], 159.04 (1C, NHCONH), 71.75 (1C, CH2O), 69.79
(1C, CH2O), 69.51 (1C, CH2O), 66.55 (1C, CH2O), 64.47–64.33
[polymer, CH2C(O)O], ꢂ62.04 [1C, CH2C(O)O], 60.42 [1C,
CH2C(O)O], ꢂ60.25 (1C, NHCHCHS), 60.15 (1C, NHCHCH2S),
55.26 (1C, SCH), ꢂ53.31 [polymer backbone C(CH3)Br], 47.95–
47.84 (polymer, CH2SO3K), 45.15–44.83 [polymer backbone
CH2C(CH3)Br], 39.68 (1C, SCH2), 35.05 [1C, (CH3)2CC(O)O], 33.62
[1C, CH2C(O)O], 27.87 (1C, SCHCH2CH2), 27.61 (1C, SCHCH2CH2),
24.11 [1C, CH2CH2C(O)O] [polymer, CH2CH2C(O)O], 23.42–23.37
[2C, (CH3)2CC(O)O], 23.35 (polymer, CH3 backbone), ꢂ18.60 (poly-
mer, CCH3 backbone), ꢂ17.19 (polymer, CH2 backbone).
1H NMR (700.13 MHz, D2O, 298 K), (Fig. 9) d (ppm): 4.61–4.58
(dd, 1H, NHCHCHS), 4.44–4.40 (dd, 1H, NHCHCH2S), 4.26–4.01
[br polymer hump, CH2OC(O); masked by peak: 2H,
CH2OC(O)], 3.82–3.61 (m, 8H, CH2O), 3.11–3.06 (m, 1H, SCH),
3.05–2.93 (br polymer hump, CH2SO3K), 2.77 (d, J ¼ 13.20 Hz,
1H, SCHH), 2.70 (br s, 1H, SCHH), 2.41–2.37 [t, J ¼ 7.48 Hz, 2H,
CH2C(O)O], 2.20–2.06 (br polymer hump,CH2CH2SO3K), 2.05–
1.75 (br polymer hump, backbone CH2), 1.75–1.70 (sextet, 2H,
SCHCH2), 1.66–1.56 [m, 8H, 2 ꢃ CH3; 2H, CH2CH2CH2C(O)O],
1.46–1.41 [quintet, 2H, CH2CH2CH2C(O)O], 1.31–0.80 (br poly-
mer hump, backbone CH3).
Synthesis of a-Biotin Poly(2-acrylamido-2-methyl-1-
propanesulfonic acid, sodium) pAAMPSA (13b)
AAMPSA (1 g, 4.36 mmol), biotin initiator (0.021 g, 0.0436
mmol), were charged in a Schlenk tube with 5 mL DMSO as
solvent. The tube was sealed with a rubber septum and
13C NMR (176.04 MHz, D2O, 298 K) d (ppm): 179.70 [1C,
CH2CH2C(O)O], 179.47 [polymer, CH2CH2C(O)O], 178.85 [1C,
SCHEME 7 Synthesis of a-biotin pSPMA (5). LRP: N-(n-Ethyl)-2-
pyridylmethanimine, CuBr, CuBr2, H2O/MeOH/DMSO, 40 ꢀC.
[Color figure can be viewed in the online issue, which is avail-
able at wileyonlinelibrary.com.]
FIGURE 8 UV absorbance spectra showing HABA analysis of
a-biotin pAAMPSA. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
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SCHEME 8 Polymerization of 2-acrylamido-2-methyl-1-propane-
sulfonic acid, sodium salt. Route a by LRP: N-(n-Ethyl)-2-pyridyl-
methanimine, CuCl, H2O/MeOH/DMSO, 30 ꢀC; Route b by LRP:
Me6-TREN, CuCl, DMSO, 25 ꢀC. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
FIGURE 11 1H NMR spectrum of pAAMPSA in D2O.
17,500 g/mol. Mn (GPC, 0.2 M NaNO3, 0.01 M NaH2PO4, PEG
calibration) ¼ 36,600 g/mol, Mw/Mn (GPC) ¼ 1.3.
Me6TREN ligand (0.023 mL, 0.0872 mmol) was injected via
a syringe. The Schlenk solution was subjected to five freeze-
pump-thaw cycles. This solution was then cannulated under
nitrogen into a second Schlenk tube, previously evacuated
and filled with nitrogen, containing Cu(I)Cl (0.0086 g, 0.0872
mmol). The temperature was adjusted to 25 ꢀC with con-
stant stirring. Samples were removed periodically using a
degassed syringe for conversion (t ¼ 0). At the end of the
polymerization, the mixture was air bubbled for 2 h. The
polymer was then precipitated twice into methanol/Acetone
(30/70), dissolved in deionized water, subsequently dialyzed
(Spectra/Por 6 standard, regenerative cellulose membrane,
MWCO 8 kDa) for 3 days and freeze-dried. 1H NMR (D2O):
The conversion was calculated by comparing the integral of
the OCAHNAC(CH3)2ACH2ASO3Na group signals of the
polymer against the monomer during polymerization. Con-
version ¼ 72% after 6 h of polymerization, DPn ¼ 74, Mn ¼
1H NMR (700.13 MHz, D2O, 298 K) d (ppm) 4.60–4.58 (dt,
1H, NHCHCH2S), 4.42–4.40 (dd, 1H NHCHCHS), 4.27–4.24
[m, 2H, CH2OC(O)], 3.79–3.64 (m, 8H, CH2O), 3.55–3.34 (br
polymer hump CH2SO3Na), 3.02–2.95 (m, 3H, SCH, SCH2),
2.39–2.37 [t, J ¼ 7.48 Hz, 2H, CH2C(O)O], 2.26–1.95 [br poly-
mer hump, polymer backbone; s, (masked by polymer peak)
6H, 2 ꢃ CH3], 1.79–1.28 [br polymer hump, (O)CNHC2(CH3);
completely masked by polymer peak: 2H,SCHCH2], 1.21–1.10
[m, 4H, CH2CH2C(O)O; CH2CH2CH2C(O)O].
13C NMR (176.04 MHz, D2O, 298 K) d (ppm): 176.27–175.47
[1C, CH2CH2C(O)O, polymer, CH2CH2C(O)O], 1C, OC(O)C
(CH3)2, 159.55 (1C, NHCONH), 71.67–68.45 (6C, CH2O),
62.03 (1C, NHCHCHS), 60.39 (1C, NHCHCH2S), 60.25 (1C,
SCH), 58.22 (CH2SO3Na), 55.25 (polymer backbone CH2Br),
52.59–52.32 [polymer (CH3)2CNH], 43.61–43.36 (polymer
FIGURE 10 13C NMR spectrum of pSPMA in D2O.
FIGURE 12 13C NMR spectrum of pAAMPSA in D2O.
BIOTIN FUNCTIONALIZED POLY(SULFONIC ACID)S FOR BIOCONJUGATION, SLAVIN ET AL.
1171

JOURNAL OF POLYMER SCIENCE PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
SUMMARY
backbone CH3CH2Br), 42.49 [O(O)CC(CH3)2], 42.36 (1C,
SCH2), 39.67 [SCH(CH2)3CH2], 33.57 (SCHCH2), 27.82
[SCH(CH2)CH2], 27.57 [CH2CH2C(O)O], 26.44–26.16 [polymer
(CH3)2CNH], 24.07 [O(O)C(CH3)2].
In summary, a-Biotin sulfonic acid polymers have success-
fully been polymerized via LRP. The biotin end group was
used to bind with an Avidin surface and QCM-D data indi-
cates the success of this. UV results of the HABA test provide
further evidence of the biotin–avidin interaction.
GPC
For aqueous GPC, the system was equipped with two PL
Aquagel-OH 8 lm mixed columns (300 ꢃ 7.5 mm) and a
guard column, with differential refractive index detection
using water (containing 0.2 M NaNO3, 0.01 M NaH2PO4) at
1.0 mL/min as the eluent. Poly(ethylene glycol) (PEG) stand-
ards (1.6 ꢃ 105–1.0 ꢃ 103 g/mol) were used for calibration.
The authors thank EPSRC, Unilever PLC (SS), and Syngenta
(ADC) for funding and John Booth for his knowledgeable dis-
cussions regarding QCM-D. Equipment used in part was sup-
ported by the Innovative Uses for Advanced Materials in the
Modern World (AM2), with support from Advantage West Mid-
lands (AWM) and part funded by the European Regional Devel-
opment Fund (ERDF).
QCM-D
The Q-Sense E4 System: QE 401 Electronics Unit, QCP 401
Chamber Platform, QFM 401 Flow Module with Ismatec IPC-
N Pump. Au coated quartz sensor [Frequency: 4.95 MHz 6
50 kHz; Cut: AT; Diameter: 14 mm; Thickness: 0.3 mm;
Finish: Optically polished, surface roughness of electrode
<3 nm (RMS); Electrode layer: 10–300 nm].
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