Synthesis of two hyaluronan trisaccharides

Article abstract of DOI:10.1021/ol0057075

(equation presented) The synthesis of two hyaluronan trisaccharides, methyl O-(β-D-glucopyranosyluronic acid)-(1,3)-O(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1,4)-O-β-D- glucopyranosiduronic acid and methyl O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1,4)-O-β-D- glycopyranosyluronic acid)-(1,3)-O-(2-acetamido-2-deoxy-β-D-glucopyranoside, are described. Construction of the target molecules was achieved though a combination of the phenyl sulfoxide and trichloroacetimidate glycosylation methodologies. This is the first report on the synthesis of the β-methyl derivatives, which represent the smallest fragments that incorporate all the structural features of polymeric hyaluronan.

Full text of DOI:10.1021/ol0057075

ORGANIC
LETTERS
2000
Vol. 2, No. 9
1279-1282
Synthesis of Two Hyaluronan
Trisaccharides
Bryan K. S. Yeung, Daniel C. Hill, Maria Janicka, and Peter A. Petillo*
Department of Chemistry, UniVersity of Illinois at Urbana-Champaign,
600 South Mathews AVenue, Urbana, Illinois 61801
alchmist@natasha.scs.uiuc.edu
Received February 22, 2000
ABSTRACT
The synthesis of two hyaluronan trisaccharides, methyl O-(â-D-glucopyranosyluronic acid)-(1,3)-O-(2-acetamido-2-deoxy-â-D-glucopyranosyl)-
(1,4)-O-â-D-glucopyranosiduronic acid and methyl O-(2-acetamido-2-deoxy-â-D-glucopyranosyl)-(1,4)-O-â-D-glycopyranosyluronic acid)-(1,3)-O-
(2-acetamido-2-deoxy-â-D-glucopyranoside, are described. Construction of the target molecules was achieved though a combination of the
phenyl sulfoxide and trichloroacetimidate glycosylation methodologies. This is the first report on the synthesis of the â-methyl derivatives,
which represent the smallest fragments that incorporate all the structural features of polymeric hyaluronan.
Hyaluronan (HA) is a member of the glycosaminoglycan
superfamily of bioactive oligosaccharides that are charac-
teristically highly functionalized, linear, and anionically
charged. Structurally, hyaluronan (Figure 1) consists of a
repeating polymer of 2-acetamido-2-deoxy-D-glucosamine
(GlcNAc or N) linked â(1,4) to D-glucuronic acid (GlcUA
or U). The disaccharide repeating units are in turn bound
through a â(1,3) linkage to form the HA chain. Although
HA occurs as a high molecular weight polymer, numerous
studies have demonstrated that only short oligosaccharides
are necessary for recognition and binding by HA proteins.1
Because of the nature of these interactions, the chemical
synthesis of small HA oligomers permits detailed studies of
how structure influences function at a molecular level. In
addition, the solution conformations of small, chemically
(1) (a) Underhill, C. J. Cell Sci. 1992, 103, 293. (b) Bonnet, F.; Dunham,
D. G.; Hardingham, T. E. Biochem. J. 1985, 228, 77. (c) Tengblad, A.
Biochem. J. 1981, 199, 297. (d) Toole, B. P. Curr. Opin. Cell Biol. 1990,
2, 839. (e) Christner, J. E.; Brown, M. L.; Dziewiatkowski, D. D. J. Biol.
Chem. 1979, 254, 4624.
Figure 1. Hyaluronan and the two target trisaccharides that
represent the polymer.
10.1021/ol0057075 CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/06/2000

synthesized HA fragments are more easily characterized than
that of the native polymeric form.
Scheme 1a
Several syntheses of HA oligosaccharides have been
previously reported.2 Among these, Slaghek and Ogawa
constructed a tetrasaccharide derivative with D-glucuronic
acid at the reducing end3 as well as a series of di-, tri-, and
tetrasaccharides with N-acetyl-D-glucosamine at the reducing
end.4 However, all of the derivatives reported incorporated
a p-methoxyphenyl ether moiety at C-1 of the reducing sugar
that facilitates chain elongation but does not accurately mimic
native HA fragments. In this regard, O-methyl is a better
structural mimic of native HA because (1) O-methyl is the
smallest functional group available to minimize any potential
secondary interactions and (2) anomerization, which signifi-
cantly complicates any NMR solution study, can be elimi-
nated by capping C-1 of the reducing end sugar.
Among the â-methyl HA derivatives previously reported
are the â(1,4) disaccharide5 and a series of tetra-, hexa-, and
octasaccharides with D-glucuronic acid at the reducing end.6
This Letter describes the first report of the â-methyl HA
trisaccharides, which represent the smallest fragments in-
corporating all of the structural features of polymeric
hyaluronan.
a (a) TMSOTf, 4 Å MS, CH2Cl2, 0 to 25 °C, 3 h; (b) (1)
guanidinium nitrate, 25 °C, 45 min; (2) benzaldehyde dimethyl
acetal, p-TsOH‚H2O, CH3CN, 45 min; (c) TMSOTf, 4 Å MS,
CH2Cl2, -30 to 25 °C, 6 h.
The UNU Trisaccharide. There are three central consid-
erations in the synthesis of glycosaminoglycans: (1) the
mode of glycosylation, or formation of the glycosidic
linkages; (2) the installation of the acetamido group; and (3)
the oxidation of C-6 on the glucuronic acid precursors.
Construction of the UNU trisaccharide utilized TMSOTf-
mediated glycosylation of methyl 2,3-di-O-benzyl-6-O-(4-
methoxybenzyl)-â-D-glucopyranoside (4)5 with the trichlo-
roacetimidate donor 57 to afford the corresponding â(1,4)
disaccharide, 6 in 87% yield (Scheme 1).
Benzylidenation afforded the disaccharide acceptor, 7, in
76% yield over two steps. Condensation of 7 with imidate 8
in the presence of a catalytic amount of TMSOTf produced
the fully protected trisaccharide 9 in 84% yield.
Conversion of the TROC carbamate into the acetamido
moiety was carried out with Cd dust9 in DMF:AcOH (2:1)
followed by treatment with acetic anhydride in pyridine
(Scheme 2) to afford 10 in 86% yield. Removal of the
To form the â(1,3) linkage, we envisioned removal of the
three acetates on 6 followed by formation of the 4,6-
benzylidene. Saponification under Zemple´n conditions (Na°/
MeOH) afforded the corresponding triol; however, in a side
reaction, the N-trichloroethoxycarbonyl group (TROC) was
inadvertently converted into the corresponding methyl car-
bamate. Consequently, an alternative, milder method was
employed utilizing a basic solution of guanidinium nitrate8
and quantitatively produced the desired triol after 45 min.
This procedure is specific for deacetylation in the presence
of a TROC carbamate.
Scheme 2a
(2) (a) Takanashi, S.; Hirasaka, Y.; Kawada, M. J. Am. Chem. Soc. 1962,
84, 3029. (b) Jeanloz, R. W.; Flowers, H. M. J. Am. Chem. Soc. 1962, 84,
3030. (c) Flowers, H. M.; Jeanloz, R. W. Biochemistry 1964, 3, 123. (d)
Walker-Nasir, E.; Jeanloz, R. W. Carbohydr. Res. 1979, 68, 343. (e) Klaffke,
W.; Warren, C. D.; Jeanloz, R. W. Carbohydr. Res. 1993, 244, 171.
(3) Slaghek, T. M.; Hypponen, T. K.; Ogawa, T.; Kamerling, J. P.;
Vliegenthart, J. F. G. Tetrahedron Lett. 1993, 34, 7939.
(4) (a) Slaghek, T.; Nakahara, Y.; Ogawa, T. Tetrahedron Lett. 1992,
33, 4971. (b) Slaghek, T. M.; Nakahara, Y.; Ogawa, T.; Kamerling, J. P.;
Vliegenthart, J. F. G. Carbohydr. Res. 1994, 255, 61.
a (a) (1) Cd dust, DMF:AcOH (2:1), rt, 8 h; (2) Ac2O, pyridine,
25 °C, 1 h; (b) DDQ, CH2Cl2, 2 h; (b) (1) Na°, MeOH, 25 °C, 30
min; (2) TEMPO, NaBr, TBABr, 5% NaOCl, NaHCO3, CH2Cl2:
H2O (6:1), 20 min; (c) (1) Pd(OH)2, H2, MeOH:H2O (10:1), 18 h.
(5) Carter, M. B.; Petillo, P. A.; Anderson, L.; Lerner, L. E. Carbohydr.
Res. 1994, 258, 299.
(6) Blatter, G.; Jacquinet, J.-C. Carbohydr. Res. 1996, 288, 109.
(7) Dullenkopf, W.; Castro-Palomino, J. C.; Manzoni, L.; Schmidt, R.
R. Carbohydr. Res. 1996, 296, 135.
(8) Ellervik, U.; Magnusson, G. Tetrahedron Lett. 1997, 38, 1627.
(9) Hancock, G.; Galpin, I. J.; Morgan, B. A. Tetrahedron Lett. 1982,
23, 249.
p-methoxybenzyl ether was achieved with DDQ in CH3CN
at 0 °C and produced the corresponding alcohol 11, in 84%
yield.
Treatment of 11 with NaOMe in methanol provided 12 in
quantitative yield. The selective oxidation of the primary
1280
Org. Lett., Vol. 2, No. 9, 2000

hydroxyls to the corresponding diacid was achieved using a
catalytic amount of 2,2,6,6-tetramethyl-1-piperidinyloxy
(TEMPO) and 5% aqueous NaOCl as an oxidant.10 Finally,
hydrogenolysis with Pearlman’s catalyst (60 psi) and puri-
fication on a Sephadex G-10 size-exclusion column, followed
by preparative TLC (4:2:2:1 n-BuOH, H2O, EtOH, AcOH)
on SiO2, afforded 2 as a clear foam in 10 steps and 22%
overall yield. Complete spectral characterization of 2, includ-
Treatment of 15 with benzaldehyde dimethyl acetal and
p-toluenesulfonic acid afforded the corresponding ben-
zylidene derivatives. The desired diastereomer was purified
on silica gel to afford pure 1612 in 12% yield over five steps.
The advantage of this method lies in the single chromato-
graphic step at the conclusion of the sequence, which
essentially makes this a one-pot procedure.13
The use of the sulfoxide glycosylation methodology
developed by Kahne14 required the preparation of sulfoxide
20 (Scheme 4). Phase transfer catalysis of glycosyl bromide
17 in the presence of thiophenol afforded the corresponding
thiophenylglycoside 18 in 87% yield.15 Subsequent deacety-
lation and formation of the p-methoxybenzylidine afforded
diol 19 (88%), which was esterified with PivCl in pyridine
(92%) and then subsequently oxidized with m-CPBA to
afford sulfoxide 20 in 69% yield.
1
ing mass spectral analysis and all H and 13C assignments,
is provided in the Supporting Information.
The NUN Trisaccharide. Although the NUN trisaccharide
contains the same monosaccharide units as the UNU trisac-
charide, alternate monomer precursor units were employed
for the glycosylation steps. Of the monomer units used to
Scheme 3a
With the desired monomer building blocks in hand,
condensation of 16 and 20 in the presence catalytic trifluo-
romethanesulfonic anhydride (Tf2O) afforded â(1,3) disac-
charide 21 in 86% yield (Scheme 5). Prior to unmasking
Scheme 5a
a (a) Ceric(IV) ammonium nitrate, NaN3, CH3CN, -20 °C, 8 h;
(b) Na°, MeOH, 25 °C, 2 h; (c) benzaldehyde dimethyl acetal,
p-TsOH‚H2O, CH3CN, 25 °C, 6 h.
construct the fully protected trisaccharide, only compound
5 was used in the syntheses of both trisaccharides. Schemes
3 and 4 outline the routes to the other monomer units used
in the NUN synthesis to form the â(1,3) linked disaccharide.
Scheme 4a
a (a) Tf2O, DTBMP, CH2Cl2, 4 Å MS, -60 to 25 °C, 1.5 h; (b)
(1) LiOH‚H2O, MeOH:THF:H2O (3:2:1), 50 °C, 24 h; (2) BnBr,
NaH, DMF, 25 °C, 6 h; (c) NaBH3CN, TFA, 3 Å MS, DMF, 25
°C, overnight; (d) TMSOTf, CH2Cl2, 4 Å MS, 0 °C, 3 h.
the hydroxyl on C-4′ by a selective reduction of the 4,6-O-
p-methoxybenzylidene, it was necessary to exchange the
pivaloyl esters with benzyl ethers (22). The lower reactivity
of the C-4′ hydroxyl coupled with the steric bulk of the
a (a) PhSH, tetrabutylammonium hydrogen sulfate, 1 M Na2CO3,
CH2Cl2, 25 °C, 45 min; (b) (1) Na°, MeOH, 25 °C, 2 h; (2)
p-methoxybenzaldehyde dimethyl acetal, p-TsOH‚H2O, DMF, 50
°C, 3 h; (c) PivCl, DMAP, pyridine, reflux, overnight; (d) m-CPBA,
CH2Cl2, -78 °C, 40 min.
(10) (a) Davis, N. J.; Flitsch, S. L. Tetrahedron Lett. 1993, 34, 1181.
(b) Garegg, P. J.; Oscarson, S.; Tedebark, U. J. Carbohydr. Chem. 1988,
17, 587.
(11) Lemieux, R. U.; Ratcliffe, R. M. Can. J. Chem. 1979, 57, 1244.
(12) (a) Pozsgay, V.; Coxon, B. Carbohydr. Res. 1995, 277, 171. (b)
Pozsgay, V.; Coxon, B. Carbohydr. Res. 1994, 257, 189.
(13) Compound 15 was reported as an undesired side product in ref 12b.
(14) (a) Kahne, D.; Walker, S.; Cheng, Y.; van Engen, D. J. Am. Chem.
Soc. 1989, 111, 6881. (b) Yan, L.; Kahne, D. J. Am. Chem. Soc. 1996,
118, 9239.
Starting from tri-O-acetyl-D-glucal (13), treatment with
ceric(IV) ammonium nitrate (CAN) and sodium azide
afforded a mixture of 2-deoxy-2-azido-1-nitrate derivatives
(14).11 Conversion of the nitrate group to the corresponding
methyl glycoside was achieved with methanolic sodium
methoxide and provided 15 as a mixture of diastereomers.
Org. Lett., Vol. 2, No. 9, 2000
1281

pivaloyl ester at C-3′ precluded the formation of the â(1,4)
linkage with 5. Treatment of 22 with TFA and sodium
cyanoborohydride produced glycosyl acceptor 23 in 91%
yield, and subsequent glycosylation with imidate 5 in the
presence of catalytic TMSOTf afforded 86% of the fully
protected trisaccharide 24.
Conversion of the TROC carbamate and the azide to the
corresponding acetamido group proceeded smoothly as a one-
pot three-step process (Scheme 6). Reduction of the TROC
crude reaction mixture was subsequently treated with acetic
anhydride to afford the diacetamido trisaccharide, 25, in 90%
yield. Removal of the p-methoxybenzyl group with CAN
afforded the free alcohol. Subsequent oxidation of the alcohol
to the corresponding carboxylic acid with catalytic TEMPO
and 5% aqueous NaOCl afforded 26 in 57% yield over two
steps.10 Following aqueous workup, the crude reaction
mixture was subjected to hydrogenolysis with Pd(OH)2 and
saponification with lithium hydroxide monohydrate. The final
purification step was carried out on Sephadex G-10 using
water as an eluent and, upon lyophilization, afforded the
target trisaccharide, 3, as a white solid in 11 steps and 18%
overall yield from the monosaccharides. Complete spectral
characterization of 3, including mass spectral analysis and
a
Scheme 6
1
all H and 13C assignments, is provided in the Supporting
Information.
In summary, the first synthesis of two â-methyl trisac-
charide derivatives of hyaluronan is described. These trisac-
charides are the smallest fragments that incorporate all of
the structural features that represent the native polysaccha-
ride. Currently, NMR solution studies of both trisaccharides
are underway to probe internal hydrogen bonding and bond
mobilities across the glycosidic linkage.
a (a) (1) Cd (dust), DMF:AcOH (1:1), 25 °C, 8 h; (2) AcSH,
pyridine, 25 °C, 12 h; (3) Ac2O, pyridine, 2 h; (b) (1) CAN, CH2Cl2:
H2O (9:1), 2 h; (2) TEMPO, NaBr, TBABr, 5% NaOCl, NaHCO3,
CH2Cl2:H2O (6:1), 30 min; (c) (1) Pd(OH)2, H2 (60 atm), MeOH:
H2O (10:1), 18 h; (2) LiOH‚H2O, H2O:THF (2:1), 25 °C, 8 h.
Acknowledgment. This research was funded by the
Petroleum Research Fund, the National Institutes of Health,
and the American Heart Association.
Supporting Information Available: Experimental pro-
cedures and complete spectroscopic data for 2, 3, and all
key intermediates. This material is available free of charge
via the Internet at http://pubs.acs.org.
group with Cd (dust) in DMF:AcOH was followed by
reduction of the azide with thiolacetic acid in pyridine. The
(15) Tropper, F. D.; Andersson, F. O.; Grand-Maitre, C.; Roy, R.
Synthesis 1991, 734.
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