Synthesis of diblock copolymers consisting of hyaluronan and poly(2-ethyl-2-oxazoline)

Article abstract of DOI:10.1021/ma047439m

The preparation of biomimetic diblock copolymers consisting of two hydrophilic units which include a nonionic block (PEtOz) and an anionic block (HA) was discussed. The amine-terminated poly(2-ethtyl-2-oxazolines) (NH ₂PEtOz) was prepared using

Full text of DOI:10.1021/ma047439m

Macromolecules 2005, 38, 2043-2046
2043
groups,21,25 or alkyl chains.19 Among the various poly-
(2-alkyl-2-oxazolines), those possessing a methyl or an
ethyl side chain have attracted the most attention by
far.26-28 They are water-soluble and can be prepared
from commercially available monomers. Aqueous poly-
(2-ethyl-2-oxazoline) (PEtOz) solutions exhibit a cloud
point around 62-65 °C, depending on polymer molec-
ular weight and concentration26 and on the presence of
cosolvents and salts.27 Recently, several examples of
PEtOz-containing diblock copolymers have been de-
scribed, in which the hydrophilic PEtOz unit was linked
to a hydrophobic polymer, such as poly(isobutylvinyl
ether),29 poly(L-lactide), or poly(ꢀ-caprolactone).30 Such
amphiphilic diblock copolymers form micelles in water
and may act as delivery systems for hydrophobic
drugs.31-33
The methodology adopted in the synthesis of PEtOz-
containing diblock copolymers reported to date relies on
the sequential living polymerization of two different
monomers. This approach, however, is ill-suited to the
preparation of HA-b-PEtOz since one block, HA, is a
natural polymer. A variation of this methodology involv-
ing the enzyme-catalyzed synthesis of the polysaccha-
ride block of poly(ethylene oxide)-block-amylose was
shown to occur in good yields.34 This method could in
principle be applied to the synthesis of HA-b-PEtOz, as
Kobayashi et al. reported recently the preparation of
HA via hyaluronidase-catalyzed ring-opening polymer-
ization of the appropriate disaccharide.35 We did not
pursue it in view of the low overall yield of the elaborate
disaccharide synthesis. We opted instead to form HA-
b-PEtOz via coupling of two preformed polymers: HA
and an amine-terminated poly(2-ethyl-2-oxazoline). This
approach is viable only if the coupling reaction occurs
in high yield and can be carried out in the presence of
carboxylic acid and hydroxyl groups, the two types of
functional groups present on the HA backbone. We
opted for the reductive amination of the HA terminal
aldehyde group with a primary amine linked to PEtOz.
This reaction was found to be effective in grafting HA
to polyamines, such as poly(L-lysine).4
The chemistry used to prepare amine-terminated
poly(2-ethyl-2-oxazolines) (NH2-PEtOz) relies on the use
of the bifunctional initiator, N-[2-(p-toluenesulfonyloxy)-
ethyl]phthalimide, which was obtained in high yield
(90%) by reaction of N-(2-hydroxyethyl)phthalimide with
p-toluenesulfonyl chloride and triethylamine in chloro-
form at room temperature (see Scheme 1, Supporting
Information). It was purified by three consecutive
recrystallizations from hexane:ethyl acetate ) 3:1.36,37
The polymerization of 2-ethyl-2-oxazoline (99 mmol) was
carried out in refluxing acetonitrile using N-[2-(p-
toluenesulfonyloxy)ethyl]phthalimide as initiator (2
mmol) for a period of 24 h. The oxazolinium end groups
of the growing polymer chains were quenched by addi-
tion of methanolic NaOH to the cooled polymerization
mixture. The resulting phthalimide-terminated poly(2-
ethyl-2-oxazoline) was isolated by precipitation into
anhydrous diethyl ether, followed by 2 days dialysis
against distilled water using a Spectra/Por CE mem-
brane (molecular weight cutoff of 5000 g/mol). The
experimental number-average molecular weight (Mn )
5500 g/mol) determined by GPC, using DMF as an
eluent and a multiangle laser light scattering (MALLS)
Synthesis of Diblock Copolymers Consisting
of Hyaluronan and Poly(2-ethyl-2-oxazoline)
Yali Yang,† Kazunori Kataoka,‡ and
Franc¸oise M. Winnik*,†
Faculty of Pharmacy and Department of Chemistry,
Universite´ de Montre´al, C.P. 6128 Succursale Centre-Ville,
Montre´al, Que´bec H3C 3J7, Canada, and Department of
Materials Science, Graduate School of Engineering, The
University of Tokyo, 7-3-1 Hongo, Tokyo 113 8656, Japan
Received December 13, 2004
Revised Manuscript Received January 31, 2005
Glycoproteins are ubiquitous constituents of living
organisms where they play key functions in mechanisms
such as bioadhesion, cell-cell interactions, and recogni-
tion phenomena.1 The chemical structure of most gly-
coproteins is extremely complex and presents significant
interspecies variability. Recognizing the utility of mac-
romolecules that combine in their structure polypeptide
and polysaccharide fragments, polymer chemists have
prepared synthetic analogues of glycoproteins with
controlled composition and architecture. Examples of
such biomimetic polymers include polyoxazoline-gly-
copeptide block copolymers2,3 as well as comb-type
copolymers consisting of a poly(peptide) chain grafted
with polysaccharide chains.4 These polymers have found
applications as constituents of targeted drug delivery
systems and surface modifiers of various biomaterials.
We introduce here a different class of glycoprotein
analogues, where a linear polysaccharide block is linked
to a pseudopeptide block. Specifically we describe AB
diblock copolymers consisting of a hyaluronan block and
a poly(2-ethyl-2-oxazoline) block. The combination of
these two polymers offers unique possibilities, in terms
of biotechnological applications. Hyaluronan is a natural
polymer consisting of alternating N-acetyl-â-D-glu-
cosamine and â-D-glucuronic acid residues linked at the
1-3 and 1-4 positions, respectively.5 It plays a critical
role as signaling molecule in cell motility,6 wound
healing,7 cancer metastasis,8 and morphogenesis.9 Un-
modified and modified HA have found various applica-
tions in drug delivery systems and surgery.10-15 Hyalu-
ronan has been found to enhance absorption of drugs
and proteins through mucus tissues.16 It has also been
applied, together with the polycation chitosan, in the
form of polyelectrolyte multilayer coatings to create
biocompatible surfaces and to reduce the thromboge-
nicity of injured arteries.17,18
Poly(2-alkyl-2-oxazolines) are nontoxic neutral hy-
drophilic macromolecules that show excellent biocom-
patibility and have found applications as components
of drug delivery systems and in antimicrobial formula-
tions.19 They are synthesized in near-uniform chain
length by living cationic polymerization of the corre-
sponding 2-alkyl-2-oxazoline. By selecting appropriate
polymerization initiators20 and quenchers, it is possible
to synthesize poly(2-alkyl-2-oxazolines) terminated at
one end, or both, with functional groups, such as double
bonds,21 siloxanes,22 tosylates,23 hydroxyl groups,24 amine
† Universite´ de Montre´al.
‡ The University of Tokyo.
* To whom correspondence should be addressed: Tel 1-514-340-
5179; Fax 1-514-340-3245; e-mail francoise.winnik@umontreal.ca.
10.1021/ma047439m CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/12/2005

2044 Communications to the Editor
Macromolecules, Vol. 38, No. 6, 2005
Table 1. Molecular Characteristics of the Polymers
Investigated
GPC
entry
Mtnheor
Mw
Mw/Mn
block ratioe
HA
2700c
6800d
6800d
10200c
1.26
1.23
1.17
1.31
PhtN-PEtOz
NH2-PEtOz
HA-b-PEtOz
4900a
4700
9500b
1:9.9
a From the ratio of [monomer] to [initiator]. b Calculated from
GPC results of components. c Acetate buffer. d DMF. e From 1H
NMR data.
Figure 1. Gel permeation chromatograms of the diblock
copolymer HA-b-PEtOz (solid line) (Mw ) 1.02 × 104 g/mol,
Mw/Mn ) 1.31) and HA (dashed line) (Mw ) 2.75 × 103 g/mol,
Mw/Mn ) 1.26): column, Viscotek-8025, Tskgel; eluent, buffer
solution, 0.3 M acetic acid, 0.2 M sodium acetate and 0.8 mM
sodium azide (pH: 4.5); flow rate, 0.5 mL/min; temperature,
25 °C; and detection, RI and LS.
detector, was close to the value predicted on the basis
of the initial monomer/initiator ratio (Mn,calc ) 4900
g/mol) (Table 1). The 1H NMR spectrum of PhtN-PEtOz
in D2O presents two doublets at 7.58 and 7.26 ppm,
attributed to the aromatic protons of the terminal
phthalimide group. The degree of polymerization of the
polymer was calculated from the integrated areas of
these two signals and of the broad singlet at 3.46 ppm,
attributed to the methylene protons of the PEtOz
backbone. The number-average molecular weight (Mn
) 5200 g/mol) derived from integration of the 1H NMR
signals is in excellent agreement with the number-
average molecular weight determined by GPC.
We set out next to convert the terminal phthalimide
group into a primary amine group. This transforma-
tion was performed by treatment of PhtN-PEtOz with
hydrazine monohydrate in ethanol at room tempera-
ture. The success of the reaction was ascertained by
GPC/1H NMR analysis to confirm (1) that the molecular
weight of the polymer was not affected by the reaction
and (2) that the phthalimide group had been removed.
The GPC-derived number-average molecular weight of
the amine-terminated poly(2-ethyl-2-oxazoline) (NH2-
PEtOz) was nearly identical to that of PhtN-PEtOz
(Table 1). The polydispersity index (Mw/Mn) remained
low as well.38 No signal was detected in the aromatic
region in the 1H NMR spectrum of NH2-PEtOz (Figure
2b), confirming the successful removal of the phthal-
imide group.
The hyaluronan sample (Table 1) used for coupling
with the NH2-PEtOz sample was obtained by hyalu-
ronidase degradation (37 °C, 30 h, PBS buffer, pH
7.2) of a commercial HA sample (Mn 590 000 g/mol),
followed by purification by size exclusion chromatogra-
phy over a Sephadex G-75 filter to ensure narrow
polydispersity of the sample. Coupling of this HA sample
Scheme 1. Synthesis of the Diblock Copolymer Hyaluronan-block-poly(2-ethyl-2-oxazoline) (HA-b-PEtOZ) and
Structure of the Copolymer

Macromolecules, Vol. 38, No. 6, 2005
Communications to the Editor 2045
neutral segment and a positively charged segment, such
as poly(ethylene glycol)-block-poly(L-lysine).42,43 It may
also be used as a temperature-sensitive material, which
may undergo heat-induced micellization in water.44 Both
types of micelles will be tools as delivery systems, taking
advantage of the biological properties of HA. Conversely,
they will also offer new means to explore the biological
properties of HA.
Acknowledgment. This work was supported by a
strategic grant of the Natural Sciences and Engineering
Research Council of Canada (to F.M.W.) and by the
Special Coordination Funds for Science and Technology
from the Ministry of Education, Culture, Sports, Science
and Technology of Japan (MEXT) (to K.K.).
Supporting Information Available: Detailed synthetic
procedures and spectral data for all polymers and for the
initiator; synthetic scheme for the preparation of NH2-PEtOz.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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with NH2-PEtOz was performed at 40 °C in an aqueous
sodium borate buffer (pH 8.5) for a period of 8 days
in the presence of NaBH3CN (Scheme 1). Purification
of the reaction product by extensive dialysis, first
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NH2-PEtOz, gave, after lyophilization, HA-b-PEtOz in
49% yield and 90% purity. The success of the coupling
reaction and of the purification procedure was ascer-
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2046 Communications to the Editor
Macromolecules, Vol. 38, No. 6, 2005
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MA047439M