Neoglycopolymers based on 4-vinyl-1,2,3-triazole monomers prepared by click chemistry

Article abstract of DOI:10.1002/mabi.200900199

The synthesis of a new glycomonomer based on mannose, prepared via CuAAC, is reported. The resulting 1,2,3-triazole linkage between mannose and the polymer backbone ensures the formation of highly stable glycopolymers, which will not undergo hydrolysis. The monomer 2′-(4-vinyl-[1,2,3]-triazol-1-yl) ethyl-O-α-D-mannopyranoside was polymerized in the presence of a RAFT agent - 3-benzylsulfanylthiocarbonylsulfanyl propionic acid - to yield well-defined polymers with molecular weights up to 51 500 g mol^-1 and a PDI of 1.16. The resulting polymer was employed as a macroRAFT agent in the polymerization of NIPAAm in order to generate thermo-responsive block copolymers, which undergo reversible micelle formation at elevated temperatures. The rapid interaction between the polymers prepared and ConA confirms the high affinity of these structures to proteins. While the linear glycopolymers already undergo a fast complexation with ConA, the reported rates have found to be exceeded by the micellar glycopolymer structure presented in the current contribution.

Full text of DOI:10.1002/mabi.200900199

Communication
Neoglycopolymers Based on 4-Vinyl-1,2,3-
Triazole Monomers Prepared by Click
Chemistrya
Martin Hetzer, Gaojian Chen, Christopher Barner-Kowollik,
Martina H. Stenzel*
The synthesis of a new glycomonomer based on mannose, prepared via CuAAC, is reported.
The resulting 1,2,3-triazole linkage between mannose and the polymer backbone ensures the
formation of highly stable glycopolymers, which will not undergo hydrolysis. The monomer
20-(4-vinyl-[1,2,3]-triazol-1-yl)ethyl-O-a-D-mannopyranoside was polymerized in the presence
of a RAFT agent – 3-benzylsulfanylthiocarbonylsulfanyl propionic acid – to yield well-defined
polymers with molecular weights up to 51 500 g molꢀ1 and a PDI of 1.16. The resulting polymer
was employed as a macroRAFT agent in the polymerization of NIPAAm in order to generate
thermo-responsive block copolymers, which undergo reversible micelle formation at elevated
temperatures. The rapid interaction between the polymers prepared and ConA confirms the
high affinity of these structures to
proteins. While the linear glycopolymers
already undergo a fast complexation
with ConA, the reported rates have
found to be exceeded by the micellar
glycopolymer structure presented in
the current contribution.
Introduction
M. Hetzer, G. Chen, M. H. Stenzel
Glycopolymers—synthetic polymers with pendant carbo-
Centre for Advanced Macromolecular Design (CAMD), School of
hydrates—have received increasing attention not only due
Chemical Sciences and Engineering, University of New South
to their potential biocompatibility and their good water-
Wales, Sydney, Australia
solubility. Glycopolymers are even more attractive as
Fax: þ61-2-93856250; E-mail: m.stenzel@unsw.edu.au
bioactive polymers that can readily interact with their
C. Barner-Kowollik
biological environment.[1,2] The pendant sugar molecule
has a strong affinity to specific proteins, which can result in
the formation of stable complexes. An example of a
bioactive sugar molecule is mannose, which strongly binds
to Concanavalin A (ConA), a tetrameric lectin. This selective
recognition is important for cellular signaling events, such
as cell adhesion, proliferation, and survival.[3] Several
studies have been performed on the binding interactions
Preparative Macromolecular Chemistry, Institut fur Technische
¨
Chemie und Polymer Chemie, Universit¨at Karlsruhe (TH)/
Karlsruhe Institute of Technology (KIT), Engesserstrasse 18, 76128
Karlsruhe, Germany
a : Supporting information for this article is available at the bottom
of the article’s abstract page, which can be accessed from the
journal’s homepage at http://www.mbs-journal.de, or from the
author.
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DOI: 10.1002/mabi.200900199
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M. Hetzer, G. Chen, C. Barner-Kowollik, M. H. Stenzel
between ConA and polymers[4–6] containing mannopy-
ranoside repeating units.
facile synthesis procedure to generate novel glycomono-
mers based on mannose. Subsequently, reversible addition
fragmentation chain transfer (RAFT) polymerization[22]
was employed to create well-defined homo- and diblock
copolymers, which can self-assemble into micelles. A major
point of interest is the bioactivity of the prepared polymers.
As mentioned earlier, the presence of the polymeric
backbone may have a substantial influence on the selective
binding between mannose and the protein ConA.
Reflecting the rising importance of glycopolymers as
materials at the interface between polymer science and
biology is the increasing amount of synthesis approaches
reported in literature.[7,8] Glycopolymers are obtained
either by the polymerization of carbohydrates modified
with vinyl functionalities or by the postmodification of
reactivepolymersasdemonstratedonexamplessuchasthe
Cu(I) catalyzed alkyne and azide 1,3-dipolar click cycloaddi-
tion(CuAAC), whichusesazidecontaining carbohydratesto
reacttoapolymerbackbone[9] orthethiol-eneclickreaction,
which utilizes polymers with pendant vinyl functionalities
and glucothiose, a sugar derivative with thiol function-
ality.[10,11] Most of these approaches have in common that
thefinalpolymerstructurecontainesterfunctionalitiesasa
linking moiety between the polymer backbone and the
pendant sugar group, which can potentially hydrolyze
under alkaline conditions or in the presences of hydro-
lytically active enzymes.[12]
Experimental Part
Materials
Triethylamine (ꢁ99%), vinyl bromide (98%), ethynyltrimethylsi-
lane (98%), copper(I) iodine, bis(triphenylphosphine)palladium(II)
dichloride (purum, ꢁ98.0%), 2-bromoethanol (95%), D-mannose
(powder, cell culture tested), sodium azide (purum p.a., ꢁ99.0%),
amberlite1 IR-120, tetrabutylammonium fluoride (1.0 M in tetra-
hydrofuran, THF), N-isopropylacrylamide (NIPAAm) (97%), 4,40-
azobis(4-cyanovaleric acid) (ꢁ75%) were purchased from Sigma-
Aldrich and used as received. Technical grade diethyl ether, sodium
hydrogen sulfate, sodium hydrogen carbonate, sodium chloride,
magnesium sulfate, petrolether 40/60, tetrabutylammonium
hydrogen sulfate, magnesium sulfate, methanol, ethyl acetate,
sodium ascorbate, copper(II) sulfate, tetrahydrofuran, ethyl acet-
ate, and methanol were used without any further purification. The
RAFT agent, 3-benzylsulfanylthiocarbonyltsufanyl propionic acid,
was synthesized according to a procedure described elsewhere.[23]
The quest for the polymer chemist is to find a synthetic
procedure that is facile without taking recourse to
protective chemistry, which also results in stable glycopo-
lymers with high bioactivity. The bioactivity can be
hampered by the conjugation of carbohydrates to the
polymer backbone as has been demonstrated in the case of
mannose. While mannose attached to a polymer backbone
via the 1-position shows a strong binding activity towards
ConA, poly(6-O-methacryloyl mannose) loses its activ-
ity.[13] Computational work suggests that free hydroxyl
groups at the 3-, 4-, and 6-carbon positions of mannose
dictate the binding ability of ConA.[14]
Synthesis of 4-Trimethylsilyl-1-buten-3-yne
To a two-neck flask equipped with Dimroth condenser, 200 mL of
dry triethylamine were added. The solution was cooled in an ice
bath to 0 8C and degassed with nitrogen for 2 h prior the addition of
vinyl bromide (14.89 g, 139.22 mmol) and trimethylsilylacetylene
(7.92 g,80.64 mmol).Thecolorlessreactionmixturewasstirredfora
further 1 h at 0 8C and was subjected to a single freeze-pump-thaw
cycle before the addition of copper(I) iodine (0.169 g, 0.887 mmol)
and bis(triphenylphosphine)palladium(II) dichloride (0.257 g,
0.366 mmol). After two additional freeze-pump-thaw cycles, the
light yellow reaction mixture was stirred overnight at room
temperature and under constant nitrogen flow. Diethyl ether
(200 mL) was added to the brown suspension and the organic layer
was washed with ice cold 1 M NaHSO4 (5 ꢂ 200 mL), saturated
NaHCO3 (1 ꢂ 200 mL) and saturatedNaCl (1 ꢂ 200 mL). The ethereal
layer was dried over MgSO4, filtered, which gave a clear orange
liquid, and concentrated through evaporation, which gave a brown
crude product. The crude product was purified by column
chromatography on silica gel with a diethyl ether/petrol ether
mixture as eluent (1:1 v/v). The product solutions (Rf ¼ 0.88) were
evaporatedasmuchaspossibleandthefinalproductwasseparated
with an oil pump into a receiving flask cooled to ꢀ78 8C yielding
2.35 g (23.34%) of a colorless liquid.
As pointed out above, many glycopolymers are suscep-
tible to hydrolytic activity. To eliminate potentially labile
ester functionalities, glycomonomers based only on hydro-
lytically stable functional groups are attractive in a range of
applications. Reports on such glycomonomers, such as a
styrene based glycomonomers[15] or polymers with
attached mannose to the polymer backbone via amide
bonds,[16] are rare.
A monomer class related to styrene are C-vinyl-hetero-
aromatic monomers such as 4-vinyl-1,2,3-triazole.[17] 4-
Vinyl-1,2,3-triazole shows atypical polymerization kinetics
such as non-linear concentration dependence of the rate of
polymerization on the monomer concentration.[17] These
types of monomer were almost forgotten in recent years,
probably because of the elaborate synthesis procedure.
With the rise of CuAAC,[18,19] 4-vinyl-1,2,3-triazol mono-
mers experience a renaissance. Hawker and co-workers
developed a one-pot and a two-step synthetic procedure
based on click chemistry to generate 4-vinyl-1,2,3-tria-
zole[20] leading to an array of N-functionalized-4-vinyl-
1,2,3-triazoles at a fast reaction rate and with high
conversions.[21] As depicted in Scheme 1, we adopted this
1H NMR (CDCl3, 300 MHz): d ¼ 0.20 (s, 9H, Si(CH3)3), 5.47 (d,
J ¼ 11.1 Hz, 1H, trans CH2 ¼ CH), 5.65 (d, J ¼ 2.4 Hz, 1H, cis CH2¼CH),
Macromol. Biosci. 2010, 10, 119–126
120
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DOI: 10.1002/mabi.200900199

Neoglycopolymers Based on 4-Vinyl-1,2,3-Triazole Monomers Prepared . . .
Synthesis of Monomer
H
CuI
PdCl2(PPh3)2
H
+
Si
N
Br
NEt3
Si
H
TBAHS
H2O
Br
N
N
N3
+
HO
Na
+
HO
HO
OH
OH
OH
OH
OH
O
+
O
Amberlite IR-120
HO
HO
N3
HO
HO
OH
O
O
N3
sodium ascorbate
CuSO4
OH
OH
TBAF
OH
O
O
HO
HO
HO
HO
Si
THF:H2O 1:1
O
N
N
N3
N
Synthesis of Homo- and Block Copolymers
OH
OH
O
S
S
HO
HOOC
HO
H
n
HO
OH
ACPA, 60 °C
S
O
S
S
N
HOOC
OH
+
O
N
N
H2O:MeOH 2:1
N
S
N
N
HO
H
N
O
ACPA, 60 °C
S
S
S
S
HOOC
n
HOOC
n
m
OH
S
S
HO
OH
O
OH
O
N
HN
O
HO
HO
N
HN
H
N
N
OH
N
O
OH
O
O
H
Scheme 1. Synthesis of 20-(4-vinyl-[1,2,3]-triazol-1-yl)ethyl-O-a-D-mannopyranoside and the subsequent RAFT polymerization to prepare
thermo-responsive block copolymers.
5.82 (dd, J ¼ 17.6 Hz, 11.1 Hz, 1H, CH2¼CH). 13C NMR (CDCl3,
300 MHz): d ¼ 0.25, 95.4, 104.1, 117.6, 128.3.
ether (150 mL) was added and the organic layer was washed with
distilled H2O (3 ꢂ 20 mL). The organic phases were collected, dried
over MgSO4 and filtrated. The reaction mixture was evaporated to
drynessandtheproductfurtherdriedundervacuumyielding6.08 g
(21.03%) of a slightly yellow liquid.
Synthesis of 2-Azidoethanol
1H NMR (CDCl3, 300 MHz): d ¼ 3.78 (t, J ¼ 5.1 Hz, 2H, OꢀCH2), 3.45
2-Bromoethanol (20.76 g, 0.166 mol), sodium azide (21.58 g,
0.332 mol), and tetrabutylammonium hydrogen sulfate (1.00 g,
2.95 mmol) were added to a round bottom flask filled with 40 mL
distilled water. The reaction mixture was stirred for 2 d, giving a
white suspension with orange droplets on the surface. Diethyl
(t, J ¼ 4.9 Hz, 2H, N3ꢀCH2).
CAUTION! Although we had never experienced any adverse
events when working with these products, organic azides are
potentially explosive compounds and they should be handled with
utmost care!!
Macromol. Biosci. 2010, 10, 119–126
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121

M. Hetzer, G. Chen, C. Barner-Kowollik, M. H. Stenzel
distilled water and mixed with 0.1 mL of ACPA (0.116 mg,
4.149 ꢂ 10ꢀ4 mmol) also dissolved in water and RAFT agent
(0.226 mg, 8.298 ꢂ 10ꢀ4 mmol) dissolved in methanol. The reaction
mixture was transferred to a Schlenck tube and thoroughly
deoxygenated by three consecutive freeze-pump-thaw cycles. The
reaction mixture was then transferred into an IR-cuvette and
purged with nitrogen for a further 45 min, before being placed in
theFT-IRfora 24 hpolymerizationat60 8C. Themolecularweightof
the resulting polymer after 24 h was measured to be
Mn ¼ 58 500 g ꢃ molꢀ1 by NMR and Mn ¼ 51 500 g ꢃ molꢀ1 by GPC
with a polydispercity index (PDI) of 1.16.
Synthesis of 2(-Azidoethyl-O-a-D-mannopyranoside
The procedure was described earlier by Haddleton et al.[24]
Amberlite IR-120 (1.24 g) and 2-azidoethanol (6.08 g, 69.82 mmol)
were added to a roundbottom flask equipped with a condenserand
heated to 90 8C. After 30 min D-mannose (1.24 g, 6.9 mmol) was
added in a single portion. After the reaction mixture was stirred at
90 8C for a further 2.5 h, the suspension was filtered through a very
short cotton wool pad and the filtered solid was washed with
methanol. 3.5 g silica were added to the slightly yellow solution,
stirred for 1 h and the remaining solvent removed through
evaporation. The crude product dispersed on the silica gel was
purified by column chromatography using an ethyl acetate/
methanol mixture as eluent (19:1 v/v) to yield 0.86 g (50.6%) of a
slightly yellow powder.
Synthesis of Poly(2(-(4-vinyl-[1,2,3]-triazol-1-yl)ethyl-
O-a-D-mannopyranoside)-block-poly(N-isopropyl
acrylamide) via RAFT Polymerization
1HNMR(300 MHz,D2O):d ¼ 3.54(t,J ¼ 7.2 Hz,2H,N3ꢀCH2), 3.65–
3.99 (m, 8H), 4.92 (d, J ¼ 1.7 Hz, 1H, anomeric H).
NIPAAm (32 mg, 2.83 ꢂ 10ꢀ4 mol) and macroRAFT agent (9 mg,
2.3 ꢂ 10ꢀ7 mol) were dissolved in 0.5 mL N,N-dimethylacetamide
(DMAc). A stock solution of 2.95 mg 2,20-azoisobutyronitrile (AIBN)
in 2 mL N,N-dimethylformamide (DMF) was prepared. 20 mL of this
solution was added to the monomer macroRAFT agent mixture to
yield a concentration ratio of [NIPAAm]:[macroRAFT]:[AIBN] ¼
1 200:1:0.5.Thevialwassealedandthoroughlydegassedviafreeze-
pump thaw and heated for 24 h at 60 8C. The mixture was then
dialysed against distilled water (molecular weight cut-off 10 kDa)
and freeze-dried. The molecular weight of the resulting polymer
was measured to be Mn ¼ 32 400 g ꢃ molꢀ1 (PDI ¼ 1.12). From NMR,
the composition of the polymer was calculated to be poly-
Synthesis of 2(-(4-Vinyl-[1,2,3]-triazol-1-yl)ethyl-O-a-
D-mannopyranoside
20-Azidoethyl-O-a-D-mannopyranoside (0.73 g, 2.93 mmol), sodium
ascorbate (0.0960 g, 0.485 mmol), CuSO4 ꢃ 5 H2O (0.0583 g,
0.234 mmol), 4.4 mL of tetrabutylammonium fluoride (1 M in
THF) and 4-tetramethylsilyl-1-buten-3-yne (0.47 g, 3.73 mmol)
were weighed into a small round bottom flask, which was filled
up with 4.4 mL of distilled water. The flask was fitted with a rubber
septum and the solution was allowed to stir for 2 d. The now dark
brown reaction mixture was transferred into a round bottom flask.
THF wasremovedthrough evaporation and theremaining solution
freeze-driedovernight.Thecrudemonomerwaspurifiedbycolumn
chromatographyonsilica gelwithethyl acetate/methanolmixture
as eluent (19:1 v/v) in order to obtain 0.332 g (36.5%) of a slightly
orange solid.
(20-(4-vinyl-[1,2,3]-triazol-1-yl)ethyl-O-a-D-mannopyranoside)129
-
block-poly(N-isopropylacrylamide)93
.
Turbidimetry Assay
1H NMR (300 MHz, D2O): d ¼ 3.6–3.72 (m, 4H, H4, H3, H6), 3.86
(m, 1H, H5)3.92(m, 1H, H2), 4.09(m, 2H, OꢀCH2ꢀCH2-triazole), 4.63
(m, 2H, OꢀCH2ꢀCH2-triazole), 4.86 (s, 1H, anomeric H), 5.42
(d, J ¼ 10.4 Hz, 1H, trans CH2¼CH), 5.90 (d, J ¼ 17.8 Hz, 1H,
cis CH2¼CH), 6.70 (dd, J ¼ 29.1 Hz, 11.3 Hz, 1H, CH2¼CH), 8.06
(s, 1H, triazole). 13C NMR (300 MHz, D2O): d ¼ 51.8 (CH2 CH2N), 62.4
(CH2CH2N), 67.3 (C4), 68.1 (C6), 71.5 (C3), 72.1 (C5), 74.5 (C2), 101.2
(anomeric C), 119.0 (CH2¼CH), 124.7 (CꢀCH2ꢀN, triazole), 126.4
(CH2¼CH), 148.0 (CꢀCH2ꢀN, triazole).
A solution of 1 mM ConA in pH 7.4 HEPES-buffered saline (HBS) was
made fresh before the assay. Turbidity measurements were
performed by adding 500 mL of the ConA solution to a dry quartz
microcuvette and put into the holder of a Cary 300 UV-VIS
Spectrophotometeratacertaintemperaturefor1 min. Asolutionof
the ligand in HBS buffer was then added (50 mL at 500 mM per
mannose residue). Upon addition, the solution was mixed
vigorously for 5 s using a pipette. Absorbance data were recorded
at 420 nm for 10 min at 1.2 Hz.
ESI-MS
(exp) ¼ 324.39;
FT-IR: 3320, 2923, 1641, 1360, 1228, 1133, 1090, 1049, 1027, 976,
917, 873, 806, 670 cmꢀ1
[C12H19N3O6Na]þ:
m/z
(theo) ¼ 324.12,
m/z
NMR Spectroscopy
.
All NMR spectrums were recorded using a Bruker 300 MHz
spectrometer. Deuterated chloroform, deuterium oxide or deuter-
ated dimethyl sulfoxide were used as solvent.
Synthesis of Poly(2(-(4-vinyl-[1,2,3]-triazol-1-yl)ethyl-
O-a-D-mannopyranoside) via RAFT Polymerization
Homopolymerisations of 20-(4-vinyl-[1,2,3]-triazol-1-yl)ethyl-O-a-
D-mannopyranoside (1.106 mol ꢃ Lꢀ1) were performed using 4,40-
azobis(4-cyanovaleric acid) (ACPA), (1.38 ꢂ 10ꢀ3 mol ꢃ Lꢀ1) as the
initiator and 3-benzylsulfanylthiocarbonylsulfanyl propionic acid
as chain transfer agent (2.77 ꢂ 10ꢀ3 mol ꢃ Lꢀ1). Typically, the
monomer (100 mg, 0.3319 mmol) was dissolved in 0.1 mL of
Fourier Transform Near-Infrared (FT-NIR)
Spectroscopy
Reaction mixtures were deoxygenated by purging with nitrogen
for 30 min. Monomer conversions were determined via online
FT-NIR spectroscopy by observing the decrease of the vinylic
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Neoglycopolymers Based on 4-Vinyl-1,2,3-Triazole Monomers Prepared . . .
stretching overtone of the monomer at v ¼ 6 165 cmꢀ1. A Bruker
IFS\S Fourier transform spectrometer equipped with a tungsten
halogen lamp, a CaF2 beam splitter and a liquid nitrogen cooled
InSb detector were used for the FT-NIR measurements. The spectra
were recorded in the spectral region of 8 000–4 000 cmꢀ1 and were
obtained from the added interferograms of 16 scans with the
resolution of 4 cmꢀ1. The conversion was determined by selecting a
linear baseline between 6 193.05 and 6 130.30 cmꢀ1. The absor-
bance was integrated between the two points and monomer to
polymer conversions were calculated via Beer-Lambert’s law.
1,2,3-triazole derivatives by an efficient and highly stereo-
selective process, the Cu(I) catalyzed click reaction between
alkynes and azides. Click reactions are generally fast
reactions frequently leading to complete conversions.
The introduction of the vinyl functionality in 4-position
can be achieved using Wittig reactions or by applying
elimination steps.[21] However, the presence of mannose
hampered the success of these reactions leading potentially
tothedestructionofthesugarmoleculeandtoabroadarray
of products. In an alternative strategy, the synthesis of
the proposed glycomonomer, 20-(4-vinyl-[1,2,3]-triazol-1-
yl)ethyl-O-a-D-mannopyranoside, was carried outusing the
procedure outlined in Scheme 1. The synthesis of
4-trimethylsilyl-1-buten-3-yne was observed to be the
most critical step. The volatility of the reactants limit the
yield of the reaction resulting in fluctuating outcomes with
yields as low as 23%. The following Cu(I) catalyzed reaction
of 4-trimethylsilyl-1-buten-3-yne with 20-azidoethyl-O-a-D-
mannopyranoside lead to a dark brown product, from
which – upon purification via column chromatography –
the final product can be isolated at high purity. The yield of
only 36% is not an indication of the inefficiency of the click
reaction, but more the result of thorough purification of
the resulting monomer with some monomer being lost on
the column. 1H-NMR analysis confirms the structure of the
monomer and the high regio-selectivity of the click reaction
as evidenced by the single peak at d ¼ 8.06 ppm correspond-
ing to the 1,2,3-triazole ring (see electronic supporting
information). The molecular weight of the monomer was
measured via ESI-MS analysis and is in close agreement
with the theoretical value (see electronic supporting
information).
Size Exclusion Chromatography (SEC)
Molecular weight distributions were determined by SEC with a
Shimadzu modular system having DMAc (0.03% w/v LiBr, 0.05%
BHT stabilizer) at 50 8C with a flow rate of 0.85 mL minꢀ1. The
system incorporated a DGU-12A solvent degasser, a LC-10AT pump
and a CTO-10A column oven and was equipped with a RID-10A
refractive index detector. Polymer Laboratories 5.0 mm bead-size
guard
column
(50 mm ꢂ 7.8 mm)
followed
by
four
˚
5
4
3
300 mm ꢂ 7.8 mm linear PL columns (10 , 10 , 10 , and 500 A)
wereusedtoseparatethesamples.Thesystemwascalibratedusing
narrow polystyrene standards ranging from 3 000 to 106 g ꢃ molꢀ1
.
Dynamic Light Scattering (DLS)
The hydrodynamic diameters (Dh) of the aggregates were
determined using a Malvern Nano-Zetasizer. The mean diameter
wasobtainedfromthearithmeticmeanusingtherelativeintensity
of each particle size. The solution (0.5 g ꢃ Lꢀ1) was prepared in
distilled water and was filtered through 0.45 mm filters before
analysis. Samples were incubated in the holder for 5 min at certain
temperatures before the instrument start to measure the particle
size.
The following quest for a suitable RAFT agent for the
controlled polymerization of the monomer synthesized
was facilitated by earlier studies on reactivity ratios of the
copolymerization of 4-vinyl-1,2,3-triazole with styrene and
methyl methacrylate, respectively.[17] The reactivity ratios
suggest that these types of monomers have reactivities
comparable to acrylates and acrylamides. Both types of
monomers were successfully polymerized using trithio-
carbonate-type RAFT agents. Therefore, 3-benzylsulfa-
nylthiocarbonylsulfanyl propionic acid was employed as
the chain transfer agent in a mixture of monomer and 4,40-
azobis(4-cyanovaleric acid) as initiator in a water/ethanol
mixture. The rate of polymerization was subsequently
followed by the declining intensity of the vinylic bond
using FT-NIR. A monomer conversion of 47% was reached
after a polymerization time of 24 h (see electronic support-
ing information). It is recommended for further block
copolymer synthesis to stop the polymerization at an early
stage to ensure a high percentage of RAFT endgroups. High
monomer conversions can lead to the formation of dead
polymer, thus the formation of bimodal distributions in the
subsequent block copolymer synthesis.
Electrospray Ionization Mass Spectrometry (ESI-MS)
ESI-MS measurements were performed on a Thermo Finnigan LCQ
Deca quadrupole ion trap mass spectrometer (Thermo Finnigan,
San Jose, CA) equipped with an atmospheric pressure ionization
source operating in the nebulizer assisted electrospray mode
which was used in positive ion mode. Monomer samples (0.1–
0.2 mg ꢃ mLꢀ1) were dissolved in 6:4 v/v mixture of methanol/
water. Mass calibration was performed using caffeine, Met-Arg-
Phe-Ala acetate salt (MRFA, Sigma-Aldrich) and Ultramark 1621
(Sigma-Aldrich) in the m/z range 195–1 822. All spectra were
acquired within the m/z range of 150–2 000 with a spray voltage of
5 kV, a capillary voltage of 39 V, and a capillary temperature of
275 8C. Nitrogen was used as a sheath gas (flow: 40% of maximum)
and helium was used as the auxiliary gas (flow: 5% of maximum).
The instrumental resolution of the employed experimental set-up
is 0.2 amu.
Results and Discussion
Several pathways are available to generate effectively 4-
vinyl-1,2,3-triazole monomers.[20,21] Key is the formation of
Macromol. Biosci. 2010, 10, 119–126
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123

M. Hetzer, G. Chen, C. Barner-Kowollik, M. H. Stenzel
conv. 33%
Mn(GPC)= 25800 g mol-1
conv. 17%
Mn(GPC)) = 8900 g mol-1
PDI = 1.21
PDI= 1.24
Block copolymer
conv. 48%
Mn(GPC)) = 51500 g mol-1
PDI = 1.16
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
log(MW)
Figure 1. Molecular weight versus conversion of the polymerization
of 20-(4-vinyl-[1,2,3]-triazol-1-yl)ethyl-O-a-D-mannopyranoside in
water/methanol in the presence of 3-benzylsulfanylthiocarbonyl-
Figure 2. GPC curves of poly(20-(4-vinyl-[1,2,3]-triazol-1-yl)ethyl-
O-a-D-mannopyranoside) and poly(20-(4-vinyl-[1,2,3]-triazol-1-
yl)ethyl-O-a-D-mannopyranoside)129-block-poly(N-isopropyl acryl-
amide)93 prepared via RAFT polymerization.
sulfanyl propionic acid as RAFT agent. [M]0 ¼ 1.106 mol ꢃ Lꢀ1
,
[ACPA]¼ 1.38ꢂ 10ꢀ3 mol ꢃ Lꢀ1, [RAFT]¼ 2.77 ꢂ 10ꢀ3 mol ꢃ Lꢀ1
.
The living characteristic of the polymerization was
validated by confirming the linear relationship between
conversion and molecular weight. Separate samples were
prepared and isolated at a preset reaction time. The
molecularweightwasobservedtoincreasewithconversion
in close proximity to the calculated values. Deviations from
the theoretical molecular weight are assigned to the
polystyrene calibration of the GPC system (Figure 1).
In addition, the molecular weight distributions were
found to be narrow without any shoulder formation or
significant low molecular weight tailing, which would be
indicative of substantial termination reactions (see
Figure 2). It can therefore be concluded that trithiocarbo-
nates can control the polymerization of 4-vinyl-1,2,3-
triazole monomers effectively.
copolymers. A glycopolymer based macroRAFT agent with
the molecular weight of 25 800 g ꢃ molꢀ1 was therefore
polymerized in the presence of NIPAAm in DMA to yield a
block copolymer with a number average molecular weight
of 32 400 g ꢃ molꢀ1. As displayed in Figure 2, the resulting
block copolymer was found to have a symmetric molecular
weight distribution with a PDI of 1.12. The thermo-
responsive block copolymer was further investigated using
DLS. Both blocks are fully water-soluble at room tempera-
ture displaying therefore with 10.1 nm only the size of a
unimers (a single block copolymer) in the DLS analysis. At
the elevated temperature of 40 8C above the lower critical
solution temperature (LCST) of NIPAAm (32 8C), the block
copolymer takes on an amphiphilic character resulting in
the formation of micelles indicative of the increased
hydrodynamic diameter Dh of 22.5 nm (see the supporting
information). Upon cooling of the solution, the micelles
start to dissociate again.
The presence of the RAFT endgroup can be verified via
chain extension and UV/Vis analysis after intense purifica-
tion of the polymer using dialysis. It should be noted here
that the RAFT endgroup can potentially undergo hydrolysis
in aqueous solution at elevated pH values.[25] Suitable pH
values for the dialysis of RAFT polymers can be obtained by
the presence of CO2 in the water. The presence of RAFT
endgroups were also confirmed via UV/Vis analysis
employing the peak maximum at 305 nm, which correlates
to the pꢀpꢄ absorption of the yellow RAFT agent. The
UV/Vis spectrum of the polymer has been compared to
the spectrum of the RAFT agent confirming the presence of
the RAFT group as a terminal unit at each polymer chain.
The existence of the RAFT endgroup is the key to the
successful formation of block copolymers.[26] Poly(20-(4-
vinyl-[1,2,3]-triazol-1-yl)ethyl-O-a-D-mannopyranoside)
prepared via RAFT polymerization was chain extended
using NIPAAm in order to prepare thermo-responsive block
The purpose of the current study is the development of a
stable glycopolymers with pendant sugar groups of high
bioactivity. As mentioned earlier, the presence of the
polymer backbone can hamper the binding of mannose to
ConA. Mannose containing compounds are known to
prevent the clustering of red blood cells, which is caused
by the strong binding between surface carbohydrates on
these cells and ConA, since mannose will compete for the
binding sites on ConA.[27] While the binding between one
mannose molecule and ConA is rather weak, the combined
effect of multiple mannose ligands increases the affinity to
ConA significantly. It is not only the amount of mannose
that determines the binding to ConA. The affinity to ConA
can furthermore be enhanced by carefully chosen polymer
architectures.[27] To test the binding ability of the
Macromol. Biosci. 2010, 10, 119–126
124
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/mabi.200900199

Neoglycopolymers Based on 4-Vinyl-1,2,3-Triazole Monomers Prepared . . .
0.16
0.14
Conclusion
Block copolymer (40oC)
In conclusion, we demonstrated in this communication
that click chemistry is not only a versatile tool for the design
of complex polymer architectures, but it can also be
employedtogeneratebioactiveandstableglycomonomers.
The resulting glycomonomers can be polymerized via RAFT
using a RAFT agent that creates an intermediate radical of
medium stability such as trithiocarbonates. The resulting
polymers bind readily to proteins, therefore the binding
ability is not hampered by the backbone.
0.12
0.10
Glycopolymer (40oC)
0.08
Block copolymer (20oC)
Glycopolymer (20oC)
0.06
0.04
0.02
0.00
0
2
4
6
8
10
12
14
time / min
Acknowledgements: M. H. S. and C. B.-K. acknowledge the
Australian Research Council (ARC) for generous funding.
The authors thank Dr. Sadik Amajjahe for helpful discussions.
The authors thank Mr. Edgar Wong for the ESI-MS measurements
and Mr. Steve Jacenyik for the excellent management of CAMD.
M. H. S acknowledges the University of New South Wales (UNSW)
for financial support. C. B.-K. acknowledges funding from the
Karlsruhe Institute of Technology (KIT) in the context of the
Excellence Initiative for leading German universities. M. H. thanks
the Dr. Jost Henkel Stiftung for a scholarship.
Figure 3. Turbidity measurements via UV/Vis spectroscopy
to monitor the cluster formation between poly(20-(4-vinyl-
[1,2,3]-triazol-1-yl)ethyl-O-a-D-mannopyranoside)129
or
poly-
(20-(4-vinyl-[1,2,3]-triazol-1-yl)ethyl-O-a-D-mannopyranoside)129
-
block-poly(N-isopropyl acrylamide)93 and ConA at different
temperatures.
synthesized glycopolymer to ConA, a solution of polymer
was mixed with ConA. With the interaction between ConA
and the glycopolymer big clusters are formed, which will
significantly scatter light. The subsequent turbidity of the
solution – monitored via UV/Vis spectroscopy – is an
indication of occurring binding events. As seen in Figure 3,
thebinding ofthehomopolymertoConAtakes placewithin
a few seconds. Elevated temperature accelerated the
reaction slightly. Interestingly, the reaction rate of the
block copolymer at 40 8C is faster than the block copolymer.
Therateandturbidityisexceedingalltheotherstructures.It
needs to beconsidered that at40 8C, whichisabove theLCST
of PNIPAAm, the formation of micelles occurs. As a result,
the glycopolymer in the micelle takes on a more brush-like
conformation, while unimeric chains in contrast are
expected to take on a coil structure. It has been reported
earlier that rigid scaffolds for sugars are entropically
favorable to enhance binding.[28] The binding between a
mannose containing polymer and ConA can be reversed by
adding excess 1-methyl-D-mannopyranoside to the solu-
tion, which acts as a competitive ligand.[29] 1-methyl-D-
mannopyranoside was therefore added to the turbid
solution of poly(20-(4-vinyl-[1,2,3]-triazol-1-yl)ethyl-O-a-D-
mannopyranoside) and ConA. An immediate decline in
turbity was observed confirming that binding between
polymer and ConA was specifically caused by the presence
of mannose (see ESI). The specific interaction between the
glycopolymer prepared and ConA was in addition tested by
using PNA, alectin fromArachisHypogaea, whichis usually
selective to galactose. Mixing polymer and PNA does not
result in increased turbity, interaction between the
polymer and other proteins is therefore absent (see ESI).
Received: June 5, 2009; Revised: July 10, 2009; Published online:
September 3, 2009; DOI: 10.1002/mabi.200900199
Keywords: click chemistry; glycopolymer; micelles; reversible
addition fragmentation chain transfer (RAFT)
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