Chemoenzymatic synthesis of neuraminic acid containing C-glycoside polymers

Article abstract of DOI:10.1021/ol027560i

(Matrix presented) Two neuraminic acid-based, C-glycoside polymers were synthesized. Preliminary studies on one of these polymers showed potent neuraminidase inhibitory activity, suggesting potential utility as an antipathogenic surface coating for the pr

Full text of DOI:10.1021/ol027560i

ORGANIC
LETTERS
2003
Vol. 5, No. 8
1187-1189
Chemoenzymatic Synthesis of
Neuraminic Acid Containing C-Glycoside
Polymers
,†
Qun Wang,† Jonathan S. Dordick,‡ and Robert J. Linhardt*
DiVision of Medicinal and Natural Products Chemistry and Department of Chemical
and Biochemical Engineering, The UniVersity of Iowa, Iowa City, Iowa 52242, and
Department of Chemical Engineering, Rensselaer Polytechnic Institute,
Troy, New York 12180
robert-linhardt@uiowa.edu
Received December 26, 2002
ABSTRACT
Two neuraminic acid-based, C-glycoside polymers were synthesized. Preliminary studies on one of these polymers showed potent neuraminidase
inhibitory activity, suggesting potential utility as an antipathogenic surface coating for the preparation of antimicrobial biomaterials.
There is growing demand for biomaterials capable of binding
and killing harmful microorganisms.1,2 In nature, glycopro-
teins, such as mucins, form a biofilm on the surface of
epithelial cells, providing a protective barrier by binding
microorganisms. The carbohydrate moieties in mucins rep-
resent 50-90% of their molecular weight, and consist of a
large number of small sialylated glycans, attached through
O-glycosidic linkages to the serine and threonine residues
of their polypeptide backbone.3 These multiple glycan chains
result in tight binding to microbial receptors through mul-
tivalent interactions.4 Studies show that low molecular weight
salivary mucin MG2 (MW ∼20 000), which contains a
relatively homogeneous collection of oligosaccharides, mainly
sialylated disaccharides and trisaccharides,5 binds to a wide
variety of microorganisms. In contrast, high molecular weight
(∼400 000) salivary mucin, MG1, which has a divergent
collection of glycans, binds to limited sites on microorgan-
isms. The differences between these two natural mucins
suggest that synthetic mucin-like polymers with homoge-
neous neuraminic acid pendants might be useful as protective
layers to bind pathogens.
Neuraminic acid (Neu5Ac) is an abundant sugar in
glycoproteins and acts as an attachment site for many
pathogens. These glycoproteins are susceptible to cleavage
by neuraminidase, an exoglycosidase that is found in virus,
bacteria, parasites, and mammalian cells. Virus neuramini-
dase releases progeny virus particles from the surface of
erythrocytes and facilitates passage of the virus through the
mucin layer covering the epithelial cell surface. In some
pathogenic bacteria, neuraminidase also acts as a virulence
† The University of Iowa.
‡ Rensselaer Polytechnic Institute.
(1) Tiller, J. C.; Liao, C.-J.; Lewis, K.; Klibanov, A. M. PNAS 2001,
98, 5981.
(2) Tiller, J. C.; Lee, S. B.; Lewis, K.; Klibanov, A. M. Biotechnol.
Bioeng. 2002, 79, 465.
(3) (a) Mouricout, M.; Vedrine, B. Lectins Pathol. 2000, 157. (b) Chance,
D.; Reilley, T.; Smith, A. J. Microbiol. Methods 2000, 39, 49. (c) DeSouza,
M.; Surveryor, G.; Price, R.; Julian, J.; Kardn, R.; Zhou, X.; Gendler, S.;
Hilkens, J.; Carson, D. J. Reprod. Immunol. 1999, 45, 127. (d) Sheehan, J.
K.; Thornton, D. J.; Somerville, M.; Carlstedt, I. Am. ReV. Respir. Dis.
1991, 144, S4
(5) Prakobphol, A.; Tangemann, K.; Rosen, S. D.; Hoover, C. I.; Leffler,
(4) Gendler, S. J. J. Mammary Gland Biol. Neoplasia 2001, 6, 339.
H.; Fisher, S. J. Biochemistry 1999, 38, 6817.
10.1021/ol027560i CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/20/2003

factor by uncovering toxin binding sites.6 Inhibition of
neuraminidase has afforded potential strategies for the
development of antiviral and antibacterial agents.7,8 Two
neuraminidase inhibitors, Zanamirvir (Relenza, Glaxo Smith-
Kline) and Oseltamivir (Tamiflu, Hoffman-La Roche), are
currently used clinically as prophylactic agents against
influenza.9
Scheme 1a
Several groups have developed inhibitors of the influenza
virus that are polyvalent in Neu5Ac.10 The synthetic random
copolymers incorporating Neu5Ac C-glycoside moieties have
been shown to be effective in inhibiting influenza virus-
induced agglutination of erythrocytes in vitro.11 Our labora-
tory has extensive experience in the chemoenzymatic syn-
thesis of synthetic polymers containing sugar pendents.12 We
have also been particularly interested in the synthesis and
biological evaluation of neuraminic acid containing mol-
ecules.13 The current study is focused on developing the
highly efficient synthesis of catabolically stable neuraminic
acid based C-glycoside polymers for investigation as poten-
tial biomaterials for application as antimicrobial barriers.
Two such polymers were generated in this study, both
based on the initial formation of C-glycosides of neuraminic
acid. The first route involves the enzymatic polymerization
of a phenolic C-glycoside with soybean peroxidase. The
second route involves the vinyl polymerization of a terminal
allyl group on the aglycone.
C-Glycosylation donor, peracetylated neuraminic acid
phenyl sulfone (1), was synthesized according to a published
procedure.14 Samarium iodide mediated C-glycosylation
chemistry was utilized to construct a C-C bond connection
between the neuraminic acid donor and an acceptor bearing
the ketone moiety.15 Compound 3 represents a Neu5Ac
C-glycoside linked to a phenolic aglycone (Scheme 1).16
Carefully designed protecting group chemistry in the donor
and the acceptor simplified monomer synthesis. Simultaneous
deprotection of the acetyl groups and hydrolysis of the methyl
ester afforded the water-soluble phenolic C-glycoside mono-
mer (4). Both 3 and 4 were racemic (R,S) mixtures at the
newly formed hydroxylmethylene glycosidic carbon. Horse-
a Reagents and conditions: (a) THF, SmI2 10 h. (b) NaOMe,
MeOH, 8 h, followed by 0.2 M KOH. (c) Soy Bean peroxidase,
H2O2, sodium phosphate buffer, pH 7.0, 18 h.
radish peroxidase (HRP) and Soybean peroxidase (SBP) are
able to catalyze C-C bond formation, at the ortho-position
of the electron donating group-containing phenolic mono-
mers, under mild conditions. Polymerization of C-glycoside
4 in aqueous sodium phosphate buffer (pH 7) with soybean
peroxidase (SBP) and H2O2 afforded a mucin-like polymer
5 having a molecular weight of 20 000.17 Molecular weight
was determined by gel permeation chromatography and
calculated based on a standard curve with use of polysac-
charide standards. The molecular weight of polymer 5 is
similar to that of MG2 mucin (MUC 7).
(6) Corfield T. Glycobiology 1992, 2, 509.
(7) Varghese, J. N. Drug DeVelop. Res. 1999, 46, 176.
(8) Johnston, S. L. Virus Res. 2002, 82, 147.
(16) To prepare methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-
3,5-dideoxy-2-C-[(2-hydroxy-butylphenyl-acetate) methyl]-D-erythro-L-
manno-nononate 3, Neu5Ac phenyl sulfone 1 (100 mg) and 5 equiv of 4-(3-
oxobutyl)phenyl acetate 2 were dried together under high vacuum for 4 h.
SmI2 (4 equiv, freshly prepared from Sm and ICH2CH2I, 0.1 M in THF)
was added under argon in one portion at room temperature with vigorous
stirring. The dark blue solution was stirred overnight. The reaction mixture
was diluted with ether and extracted with 1 N HCl, saturated Na2S2O3,
saturated NaHCO3, and brine solution. The organic layer was dried over
anhydrous Na2SO4. The filtrate was concentrated under reduced pressure
and purified on a silica gel column with EtOAc as eluent. The C-glycoside
was obtained as oil in 82% yield. 1H NMR (CDCl3) δ 1.80-2.20 (19H,
6COCH3, H-3ax, CH2CH2Ph), 2.45 (m, 1 H, H-3eq), 2.63-2.67 (ddd, 1H,
CH2Ph), 2.78-2.85 (ddd, 1H, CH2Ph), 3.76 (d, 3H, COOCH3), 3.95-4.10
(m, 3 H, H-5, H-6, H-9a), 4.34 (m, 1 H, H-9b), 4.74 (m, H-4), 5.22 (m, 1
H, NH), 5.8 (m, 1 H, H-7), 5.40 (m, 1 H, H-8). HRFABMS: calcd for
C32H43NO15Na [M + Na]+ 704.2556, found m/z 704.2530 [M + Na]+
(17) To prepare phenolic mucin analogue 5, fully deprotected monomer
4 (10 mg, 0.022 mmol) was dissolved in sodium phosphate buffer (50 mM,
pH 7, 200 µL) 80 vol % in CH3CN. Soybean peroxidase (1 mg) was added
to the solution, followed by the dropwise (10 µL/4 min) addition of H2O2
(100 µL 0.2 M). After 12 h, the reaction mixture was freeze-dried and
subjected to BioGel P-10 column to determine molecular weight. 1H NMR
of resulting polymer shows broad peaks in the phenolic and carbohydrate
regions. The intrinstic viscosity is 2.5 × 10-3 m3/kg.
(9) Johnson, S. L. Virus Res. 2002, 82, 147.
(10) (a) Reuter, J. D.; Myc, A.; Hayes, M. M.; Gan, Z.; Roy, R.; Qin,
D.; Yin, R.; Piehler, L. T.; Esfand, R.; Tomalia, D. A.; Baker, J. R., Jr.
Bioconj. Chem. 1999, 10, 271. (b) Zanini, D.; Roy, R. J. Am. Chem. Soc.
1997, 119, 2088. (c) Wu, W.; Jin, B.; Krippner, G. Y.; Watson, K. G. Bioorg.
Med. Chem. Lett. 2000, 10, 341. (d) Choi, S.-K.; Mammen, M.; Whitesides,
G. M. J. Am. Chem. Soc. 1997, 119, 4103.
(11) (a) Nagy, J. O.; Bednarrski, M. D. Tetrahedron Lett. 1991, 32, 3953.
(b) Sparks, M. A.; Williams, K. W.; Whitesides, G. M. J. Med. Chem. 1993,
36, 778.
(12) (a) Dordick, J. S.; Linhardt, R. J.; Rethwisch, D. G. Chemtech 1994,
24, 33. (b) Wang, Q.; Linhardt, R. J.; Dordick, J. S. Biotechnol. Tech. 1999,
13, 463. (c) Wang, P.; Martin, B. D.; Parida, S.; Rethwisch, D. G.; Dordick,
J. S. J. Am. Chem. Soc. 1995, 117, 12885. (d) Wang, Q.; Dordick, J. S.;
Linhardt, R. J. Chem. Mater. 2002, 14, 3232.
(13) (a) Vlahov, I. R.; Vlahov, P. I.; Linhardt, R. J. J. Am. Chem. Soc.
1997, 119, 1480. (b) Wang, Q.; Wolff, M. W.; Polat, T.; Du, Y.; Linhardt,
R. J. Bioorg. Med. Chem. Lett. 2000, 10, 941.
(14) Mazza, A.; Sinay¨, P. Carbohydr. Res. 1989, 187, 35.
(15) (a) Curran, D. P.; Fevig, T. L.; Jasperse, C. P.; Totleben, M. J. Synlett
1992, 943. (b) Molander, G. A.; Harris, C. R. Chem. ReV. 1996, 96, 307.
(c) Krief, A.; Laval, A. Chem. ReV. 1999, 99, 745. (d) Steel, P. G. J. Chem.
Soc., Perkin Trans. 1 2001, 2727.
1188
Org. Lett., Vol. 5, No. 8, 2003

Polymer 5 was investigated as a neuraminidase inhibitor
with use of an assay commonly applied to simple Neu5Ac-
C-glycosides.18 Specifically, Ki values were determined by
incubation of the fluorescent substrate, 2′-(4-methylumbel-
liferyl)-R-D-N-acetylneuraminic acid, C. perfringens neuramini-
dase, and sodium acetate buffer at pH 5 in the presence of
various concentrations of 5. The results were fit to different
inhibition models, with the best fit obtained for a competitive
inhibition model giving a Ki of 900 nM based on the
concentration of polymer. The observed Ki was over 10-
fold lower than simple Neu5Ac-C-glycosides with hydro-
phobic aglycones.13b This enhanced inhibitory activity is
ascribed to the multivalency of the polymeric inhibitor, which
likely results from an increase of the local concentration of
the inhibitor. The synthesis of this neuraminic acid-based
polymer occurs under mild conditions, using an enzymatic
reaction, and may be feasible under physiological conditions.
Thus, a stable mucin-like polymer might be synthesized in
situ (at the surface of a wound) by application of 4 or a
suitable derivative.
Scheme 2a
Next, we examined the potential utility of such synthetic
materials as protective layers against pathogen infection.
Preliminary studies showed that mucin analogue 5 could be
incorporated on biomaterials through simple coating tech-
niques. Treatment of polystyrene beads (20-50 mesh) with
polymer 5 resulted in its binding at a high loading capacity
(2.9 mg of 5/mL of polystyrene resin), presumably through
hydrophobic interactions. The resulting activated surface is
currently being evaluated for its ability to inhibit pathogen
infection. Modification of polymer 5 was next undertaken
in an attempt to prepare higher molecular weight glycopro-
teins that resemble most natural mucins.19 The use of multiple
monomers or cross-linkers can often facilitate the enzymatic
preparation of higher molecular weight polymers. However,
the copolymerization of 4 with arbutin (4-hydroxyphenyl-
â-D-glucopyranoside) or bisphenol A as comonomers, while
affording copolymers, did not result in higher molecular
weight mucin analogues.
a Reagents and conditions: (a) SmI2, THF. (b) NaOMe, MeOH,
1 N NaOH. (c) 2,2′-azobis-(2-amidinopropane) dihydrochloride at
70 °C.
cyclohexanone (6).20 Deprotection of 7 afforded monomer
8,21 which was polymerized with 2,2′-azobis(2-amidinopro-
pane) dihydrochloride.22 The resulting polymer 9 was
precipitated from 80% aqueous methanol to yield a highly
disperse material with a MW >20 000 making it potentially
more suitable as a synthetic mucous barrier for anti-infective
applications.
These two synthetic polymers prepared bear C-linked
Neu5Ac, a specific, lectin-binding carbohydrate unit. These
polymers are, therefore, likely capable of tight multivalent
interactions with proteins and may find use as mimics of
the natural glycans found in mucins. By applying such
polymers to surfaces, such as glass, plastics, woven and
nonwoven fibers, medical instruments, etc., it may be
possible to establish a protective layer against pathogens.
A second mucin-like polymer was prepared by chemical
polymerization (Scheme 2). Monomer 7, containing a cy-
clohexylallyl functionality, was synthesized from the C-
glycosylation of Neu5Ac peracetyl-sulfone 1 with 2-allyl
OL027560I
(18) Potier, M.; Mameli, L.; Belisle, M.; Dallaire, L.; Melancon, S. B.
Anal. Biochem. 1979, 94, 287-296.
(21) To deprotect poly(vinyl) mucin analogue monomer 8, a solution of
C-glycoside 8 (33 mg) was added to MeOH (1 mL) and NaOMe in MeOH
(0.5 M, 1 mL). The mixture was stirred overnight at room temperature,
then neutralized with of Amberlite IR-120 (H+) exchange resin, filtered,
and concentrated to dryness. The residue was purified by silica gel
chromatography eluting with 100% methylene chloride, 90% and 80%
methylene chloride in methanol. 1H NMR (D2O) (R:S 1:1) δ 1.20-1.79
(m, H-3eq, CH2 × 4 in cyclohexane), 1.94 (d, 3 H, NHCOCH3), 2.39 (m,
1 H, H-3ax), 3.35-3.80 (m, 9 H).
(22) To prepare poly(vinyl) mucin analogue 9, monomer 8 (30 mg) was
dissolved in bidistilled water (2 mL). The mixture was purged with argon
for 10 min and warmed in an oil bath up to 70 °C with continuous stirring.
The polymerization was started by addition of the initiator 2,2′-azobis-(2-
amidinopropane) dihydrochloride dissolved in bidistilled water (0.1 mL).
The reaction time was 24 h. The reaction mixture was freeze-dried and the
product was precipitated in 80% methanol. The precipitate was centrifuged
and white powder was obtained after complete drying. GPC analysis showed
that the molecular weight of 9 was greater than 20 000.
(19) (a) Marquart, M.; Jamieson, A.; Blackwell, J.; Gerken, T. Biorhe-
ology 1995, 32, 431. (b) Raju, T. S.; Davidson, E. A. Biochem. Biophys.
Res. Commun. 1994, 205, 402. (c) Baszkin, A.; Proust, J. E.; Monsenego,
P.; Boissonnade, M. M. Biorheology 1990, 27, 503. (d) Slomiany, B. L.;
Sarosiek, J.; Slomiany, A. Biochem. Biophys. Res. Commun. 1987, 783.
(20) To prepare protected allyl C-glycoside monomer 7, C-glycosylation
between Neu5Ac phenyl sulfone donor 1 and 2-allylcyclohexone 6 acceptor
was carried out according to a similar procedure described for 3. The reaction
mixture was purified on silica gel chromatography with ethyl acetate as
eluent to obtain 7 as a racemic compound (yield 45%). The ratio of two
1
diastereomers was closed to 1:1. H NMR (CDCl3) δ 1.25-1.70 (m, 8H,
CH2 × 4 in cyclohexane), 1.89 (3H, CH3CONH), 2.03-2.20 (15H,
4COCH3, H-3ax, CH2CHd), 2.45-2.56 (m, 2 H, H-3eq, CH in cyclohexane),
2.63-2.67 (ddd, 1H, CH2Ph), 2.78-2.85 (ddd, 1H, CH2Ph), 3.80 (d, 3 H,
COOCH3), 3.95-4.13 (m, 3 H, H-5, H-6, H-9a), 4.30-4.40 (m, 1 H, H-9b),
4.91 (m, 2 H, H-4, H-7), 5.31 (m, 1 H, NH), 5.23-5.31 (m, 3 H, dCH2,
H-8), 5.65-5.85 (m, 1 H, CHd).
Org. Lett., Vol. 5, No. 8, 2003
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