Synthesis of a hyaluronan neoglycopolymer by ring-opening metathesis polymerization.

Article abstract of DOI:10.1039/b301734f

A hyaluronan (HA)-derived disaccharide was synthesized bearing an n-pentenyl spacer arm, which facilitated disaccharide derivatization with a norbornene template. Subsequent ring opening metathesis polymerization of the monomer produced an HA-mimetic neog

Full text of DOI:10.1039/b301734f

Synthesis of a hyaluronan neoglycopolymer by ring-opening metathesis
polymerization†
Suri Iyer,a Shyam Rele,a Gabriela Grasa,b Steven Nolanb and Elliot L. Chaikof*ac
a Department of Surgery and Biomedical Engineering, Emory University, Atlanta, GA 30322, USA.
E-mail: echaiko@emory.edu; Fax: 1-404-727-3660; Tel: 1-404-727-8413
b University of New Orleans, Department of Chemistry, New Orleans, LA 70148, USA
c
School of Chemical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA
Received (in Cambridge, MA, USA) 12th February 2003, Accepted 29th April 2003
First published as an Advance Article on the web 30th May 2003
A hyaluronan (HA)-derived disaccharide was synthesized
bearing an n-pentenyl spacer arm, which facilitated disac-
charide derivatization with a norbornene template. Sub-
sequent ring opening metathesis polymerization of the
monomer produced an HA-mimetic neoglycopolymer of low
polydispersity.
Although a number of chemical and enzymatic routes have
been developed for the generation of multivalent ligands,1,9,10
well-defined linear oligomers have proven more difficult to
synthesize. To this end, ring-opening metathesis polymerization
(ROMP)3–5 using Grubbs catalysts11,12 has been successfully
applied to synthesize glycopolymers of defined lengths with
unique bioactivities. Moreover, an added advantage of this
methodology is its tolerance to a variety of polar functionalities,
including hydroxyl, carboxylic acid, and sulfate groups.3–5,11–13
As such, ROMP provides a facile strategy for the development
of a polynorbornene based linear glycopolymer of low poly-
dispersity bearing GlcA-b-(1–3)-GlcNAc pendant carbohydrate
residues. This is the first report describing the use of ROMP for
generating well-defined, multivalent HA-mimetic neoglyco-
polymers.
Our synthetic approach involved three major steps viz., i)
synthesis of a hyaluronan-derived disaccharide containing an n-
pentenyl spacer arm; ii) attachment of the norbornene amine
functionality to the disaccharide; and iii) subsequent ROMP of
the designed monomer. The details for the synthesis of the HA-
derived disaccharide are presented in Schemes 1 and 2,
respectively. Significantly, the potential of the n-pentenyl group
to serve as a versatile handle by transformation into various
functionalities such as aldehyde, carboxylic acid, ester,
thioether, thioester or hydroxyl group, enhances the scope and
applicability of the strategy in the design and development of
novel glycomimetics. Moreover, in contrast to the post-
synthetic modification method,4 initial incorporation of the
Multivalent interactions of sugar ligands with cell surface
receptors modulate a range of biomolecular recognition proc-
esses inducing unique cellular responses that are dramatically
different from those elicited by monovalent interactions.1–7
Characteristically, multiple copies of a monovalent glycoligand
amplify the binding affinity and specificity due to a chelating
effect.1,2 For example, blocking the binding of influenza virus to
red blood cells,1 L-selectin inhibition6 during inflammation and
chemotactic responses to bacteria7 can be facilitated and
controlled by the interaction of scaffolded multivalent ligands
with specific receptor proteins. This observation has led to the
development of a number of novel neoglycopolymers that hold
significant potential in pharmacotherapy, tissue engineering,
and molecular diagnostics.
Hyaluronan (HA, 1),8 a linear non-sulfated glycosoaminogly-
can (GAG) consisting of alternating b-(1–3) and b-(1–4) linked
glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc)
residues, influences a variety of cellular processes, such as cell
growth, wound healing, and immunological responses, as well
as cancer metastasis. Significantly, these effects are mediated
by the ability of HA to interact with specific cellular receptors
in a polyvalent fashion.8 Enhancing or limiting these responses
in a controlled manner underscores the need to generate
multivalent HA-derived ligands that provide insight into
relevant protein–carbohydrate binding events. Thus, we postu-
lated that small HA fragments on a polymer backbone may
provide a starting point for mimicking selective biological
properties normally exhibited by the native polysaccharide (Fig.
1).
Scheme 1 Reagents and conditions: a) i) TfN3, MeOH, DMAP, 25 °C, 18
h, ii) Ac2O, pyridine, 0 °C, 10 h, 75%; b) H2NNH2·AcOH, DMF, 0–25 °C,
45 min, 70%; c) anhyd. K2CO3, CCl3CN, CH2Cl2, 25 °C, 48 h, 74%; d) i)
4 Å mol. sieves, TMSOTf, 4-penten-1-ol, CH2Cl2, 0 °C, 1 h, ii) MeONa,
MeOH, 0–25 °C, 6.0 h, 80% over two steps; e) CSA, THF, C6H5CH(OMe)2,
reflux, 6 h, 75%.
Fig. 1 Design of a hyaluronan-mimetic neoglycopolymer.
Scheme 2 Reagents and conditions: a) TMSOTf, CH2Cl2, 0–25 °C, 3.5 h,
78%; b) CH3COSH, 25 °C, 24 h, 70%; c) TFA–H2O (2 : 1), CH2Cl2, 0 °C,
1 h, 86%; d) 3 M NaOH, 9 : 1 MeOH–H2O, 25 °C, 2 h, 86%; e) O3 (278
°C), then add Me2S, 278 °C–25 °C, 24 h, 85%.
† Electronic supplementary information (ESI) available: spectral data for
compound 11, glycomonomer 14 and glycopolymer [A]. See http://
www.rsc.org/suppdata/cc/b3/b301734f/
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CHEM. COMMUN., 2003, 1518–1519
This journal is © The Royal Society of Chemistry 2003

sugar residues on the polymerizable template prior to the
process of ROMP, offers a facile route to generate controlled
assembly of evenly distributed HA-based oligosaccharide
residues on the polymer backbone. Briefly, the amino group in
presence of a detergent, dodecyltrimethylammonium bromide
(DTAB), under emulsion conditions (dichloroethane–water
mixture) at 60 °C for 5 h followed by chain termination with
ethyl vinyl ether afforded the HA-glycopolymer [A] as a pale
brown spongy solid (Scheme 3).‡ The ratio of molecular
equivalents of initiator : glycomonomer used was 1 : 20. The
polymerization reaction proceeded efficiently, consuming all
the monomer (monitored by TLC). The degree of polymeriza-
tion (DP) of the resultant polymer was assessed by size-
exclusion chromatography (SEC) coupled with refractive index
(RI) and laser-light scattering (LLS) detectors. Analyses of the
glycopolymer revealed that the polydispersity index (Mw/Mn)
was 1.17. Furthermore, comparison of the integrated 1H NMR
signal of the alkene protons (d 5.24 and 5.35 ppm) to that of the
terminal phenyl ring protons (d ca. 7.25 ppm) indicated a
glycopolymer molecular weight (Mn) of 9.0 kDa (n = 15.5
glycomonomer units).
In summary, an HA-substituted norbornene glycopolymer
has been synthesized using ROMP. The present approach will
facilitate the production of HA-based compounds for use in
engineering of HA-containing tissue equivalents and pharmaco-
therapy directed at modulating events mediated by HA binding
receptors.
This work was funded by grants from the NIH and the NSF
sponsored Georgia Tech/Emory Center for the Engineering of
Living Tissues.
a-
-glucosamine hydrochloride 2 was converted to the azide14
D
and the free hydroxyl groups were subsequently acetylated to
give 3. Hydrolysis of the anomeric acetate using hydrazine
acetate afforded 4, which was then converted to the imidate 5.
Introduction of the n-pentenyl group at the anomeric position
using TMSOTf as promoter at 0 °C afforded an anomeric
mixture (a : b) in the ratio 2 : 3 (1H NMR). Treatment of this
mixture under Zemplen conditions gave the triol 6, which was
then easily converted to the 4,6-benzylidene derivative 7 in 75%
yield. The two isomers 7a and 7b (ratio 2 : 3) were isolated in
pure form at this stage by column chromatography and the b-
isomer (7b) was used for further synthetic manipulations.
Glycosylation of acceptor 7b with the imidate donor 815
afforded the b-(1–3) linked HA disaccharide 9 in 78% yield.
Selective reduction of the azido group to acetamido function-
ality (10) was achieved using thiolacetic acid. Finally, debenzy-
lidenation in aqueous TFA and saponification in 3 M NaOH of
the protected N-acetylated glycoside yielded the deprotected b-
(1–3) linked HA disaccharide 11 containing the pendant n-
pentenyl spacer.‡ The product obtained was purified using
Sephadex LH-20 with methanol as the eluant to give a white
foam. In order to convert the terminal olefin to a four carbon
aldehyde, the n-pentenyl glycoside 11 was subjected to
ozonolysis, which gave the requisite product 12 (Scheme 2).
Reductive amination of the glycosidic aldehyde with 5-methyl-
aminobicyclo[2.2.1]hept-2-ene (13)16 using NaCNBH3 resulted
in the attachment of the polymerizable (norbornene) template
affording the monoalkylated glycomonomer 14 (Scheme 3).‡
The product was purified using Sephadex G-10 gel filtration
and lyophilized to give an off-white solid in an overall yield of
10% from 2.
Notes and references
‡ All the compounds have been characterized using spectral data (1H, 13
C
NMR, HRMSFAB, Maldi-Tof), see ESI.
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15 The compound 8 was prepared from commercially available aceto-
bromo-a-D-glucoronic acid methyl ester in 2 steps (50%) including
conversion of the C-1 acetyl to hydroxy (CdCO3–CH3CN, 70 °C, 3 h)
followed by introduction of the imidate group (dichloroethane–
CCl3CN–DBU, 0 °C, 1.5 h).
16 Reduction of 5-norbornene-2-carbonitrile (commercially available from
Aldrich as a mixture of isomers) using LiAlH4 afforded the amine 13
(yield 62%) as an endo : exo mixture.
Scheme 3 Reagents and conditions: a) MeOH, 25 °C, 3 h, then add
NaCNBH3, 25 °C, 15 h, 90%; b) DTAB (1 eq.), 15 (5 mol%), (CH2)2Cl2,
H2O, 60 °C, 5 h, 85%.
17 Catalyst 15 not only has activity comparable to that of the catalyst
Cl2(PCy3)2RuNCHPh, used by Kiessling and coworkers,3–5,7 but also
retains its functional group tolerance.12,13
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