Synthesis of hyaluronic acid oligomers using Ph2SO/Tf 2O-mediated glycosylations

Article abstract of DOI:10.1021/jo070704s

(Chemical Equation Presented) The synthesis of hyaluronic acid (HA) oligomers using a stepwise glycosylation strategy is described. This method employs protected 1-hydroxyuronic acid and 1-phenylthio glucosamine donors, both of which are activated with th

Full text of DOI:10.1021/jo070704s

Synthesis of Hyaluronic Acid Oligomers Using Ph2SO/
Tf2O-Mediated Glycosylations
Jasper Dinkelaar, Jeroen D. C. Code´e, Leendert J. van den Bos, Herman S. Overkleeft, and
Gijsbert A. van der Marel*
Leiden Institute of Chemistry, Leiden UniVersity, P.O. Box 9502, 2300 RA Leiden, The Netherlands
marel_g@chem.leidenuniV.nl
ReceiVed April 10, 2007
The synthesis of hyaluronic acid (HA) oligomers using a stepwise glycosylation strategy is described.
This method employs protected 1-hydroxyuronic acid and 1-phenylthio glucosamine donors, both of
which are activated with the Ph2SO/Tf2O activator system.
Introduction
at will. The exact requirements in terms of HA oligomer size
for TLR-4 binding and agonizing activity cannot be determined
by making use of the available HA oligomer mixtures (this
situation is reminiscent of alginate oligomer-mediated TLR-4
stimulation). Well-defined HA oligomers can be obtained
through organic synthesis, a strategy that has the advantage that
functionalized HA oligomers are within reach as well.
Since the pioneering work of Jeanloz6 in 1964, several
research groups have studied the synthesis of HA oligomers,7
but HA has received only a little attention from the synthetic
carbohydrate community compared to other glycosaminoglycan
family members, such as heparan. With the aim to prepare HA
oligomers that can be functionalized and used for TLR-4 binding
studies, we set out to develop a general synthesis protocol. Here
we report our results in the synthesis of a well defined, fully
deprotected HA trimer, tetramer, and pentamer, each with a
glucuronic acid moiety as the reducing end sugar.
Hyaluronan (hyaluronic acid, HA), a negatively charged high
molecular weight polymer assembled from â-1,3-linked-2-
acetamido-2-deoxy-D-glucose-â-(1-4)-D-glucuronic acid1 re-
peating units, is structurally the least complicated member of
the glycosaminoglycan (GAG) polysaccharide family. HA is
involved in many biological functions, including cell-migration,
proliferation, adhesion, recognition,2 tumor invasion (in par-
ticular malignant tumors which show increased levels of HA),3
and tumor inhibition.4 Small HA fragments have recently been
identified as potent activators of immunocompetent cells that
play a critical role in the development of T cell-mediated
immune responses.5 HA oligomers have been found to bind to
Toll-like receptor 4 (TLR-4), and it is thought that the
immunostimulating properties ascribed to HA oligomers are
effected through this receptor. The above-mentioned studies on
the immunological properties of HA oligomers made use of
compound mixtures containing HA oligomers differing in size
ranging from four monosaccharides to at least eight units. The
reason for this compound heterogeneity is that HA oligomers
are routinely prepared by enzymatic processing of HA polymers,
a process that cannot be controlled to such an extent that pure
HA oligomers of a distinct molecular weight can be obtained
Our synthesis strategy is based on our previous finding that
donor 1-hydroxysugars can be chemoselectively condensed with
acceptor 1-thioglycosides using Gin’s activator system for
dehydrative glycosylations (diphenyl sulfoxide/trifluoromethane-
(6) Flowers, H. M.; Jeanloz, R. W. Biochemistry 1964, 3, 123-125.
(7) (a) Slaghek, T. M.; Hyppo¨nen, T. K.; Kruiskamp, P. H.; Ogawa, T.;
Kamerling, J. P.; Vliegenthart, J. F. G. Tetrahedron Lett. 1993, 34, 7939-
7942. (b) Blatter, G.; Jacquinet, J.-C. Carbohydr. Res. 1996, 288, 109-
125. (c) Yeung, B. K. S.; Hill, D. C.; Janicka, M.; Petillo, P. A. Org. Lett.
2000, 2, 1279-1282. (d) Yeung, B. K. S.; Chong, P. Y. C.; Petillo, P. A.
Carbohydr. Res. 2002, 21, 779-865 (and references cited therein). (e) Iyer,
S. S.; Rele, S. M.; Baskaran, S.; Chaikof, E. L. Tetrahedron 2003, 59, 631-
638. (f) Lu, X. A.; Chou, C. H.; Wang, C. C.; Hung, S. C. Synlett 2003, 9,
1364-1366. (g) Palmacci, E. R.; Seeberger, P. H. Tetrahedron 2004, 60,
7755-7766. (h) Huang, L.; Huang, X. Chem. Eur. J. 2007, 13, 529-540.
(1) Meyer, K.; Palmer, J. W. J. Biol. Chem. 1934, 107, 629-634.
(2) Laurent, T. C.; Fraser, J. R. E. FASEB J. 1992, 6, 2397-2404.
(3) Knudson, C. B.; Knudson, W. FASEB J. 1993, 7, 1233-1241.
(4) Zeng, C.; Toole, B. P.; Kinney, S. D.; Kuo, J.; Stamenkovic, I. Int.
J. Cancer 1998, 77, 396-401.
(5) Termeer, C.; Benedix, F.; Sleeman, J.; Fieber, C.; Voith, U.; Ahrens,
T.; Miyake, K.; Freudenberg, M.; Galanos, C.; Simon, J. C. J. Exp. Med.
2002, 195, 99-111.
10.1021/jo070704s CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/22/2007
J. Org. Chem. 2007, 72, 5737-5742
5737

Dinkelaar et al.
SCHEME 1. Synthesis of Monomer HA Building Blocks
by addition of acceptor glycoside 3 (1 equiv). Following this
protocol (Scheme 2), trisaccharide 8 was prepared, but the
overall yield (12%) proved to be considerably lower than the
combined yield (26%) of the two separate glycosylation steps.
The difficulty here in our opinion is that base (TTBP) needs to
be introduced in both condensation steps in order to avoid acid-
mediated (due to in situ formation of TfOH) cleavage of the
benzylidene group in the glucosamine derivative. However, the
amount of base should be such that orthoester formation during
dehydrative glycosylation and oxazolidine formation upon
activation of the thioglucosamine donor is avoided. Indeed, we
could not enhance the efficiency of the one-pot procedure by
varying the amount of TTBP. We conclude that trisaccharide 8
is best prepared via two individual glycosylation steps, and that
the optimal amount of base used in both steps is 0.95 equiv
with respect to Tf2O.
Fully protected HA tetramer 11 and pentamer 13 were
prepared starting from trimer 8 as follows (Scheme 3). Depro-
tection of the levulinoyl group in 8 afforded trisaccharide 9
(hydrazine, pyridine/AcOH). Activation of thioglucosamine 6
using the protocol described above (Ph2SO, Tf2O, TTBP)
followed by addition of acceptor trisaccharide 9 led to the
formation of fully protected HA tetramer 11. Quenching the
reaction at -15 °C improved the yield considerably with respect
to quenching at 0 °C, and tetramer 11 was isolated in 62% yield.
In a similar fashion, but with phenylthiodisaccharide 7 as the
donor, fully protected pentasaccharide 13 (42% yield) was
prepared. Finally, the fully deprotected target tri-, tetra-, and
pentamers 10, 12, and 14 were obtained (Scheme 3) by acid
cleavage of the benzylidene group followed by saponification
of the ester and amide functionalities with KOH, N-acetylation
in MeOH with Ac2O, and purification by gel filtration.
In conclusion, we have demonstrated that HA oligomers that
are suitably functionalized for future biological studies can be
conveniently prepared by making use of thioglycosides and
1-hydroxyglycosides, in combination with the Ph2SO/Tf2O/
TTBP activating system. The yields in the glycosidic bond
formations are moderate, and the combination of the presence
of acid-labile benzylidene protective groups and the propensity
of orthoester formation requires that the glycosylations be
monitored with care, especially with respect to the amount of
base used. However, we believe our general strategy to be
convenient, in that useful quantities can be prepared from readily
available building blocks. By making use of glucuronic acid
building blocks, postglycosylation manipulations can be kept
to a minimum. We are therefore confident that our strategy will
be of use for the large-scale synthesis of HA oligomers,
including compounds with a glucosamine residue at the reducing
end and oligomers assembled from more than five monosac-
charide residues. These compounds in turn will prove to be
useful in the assessment of the biological properties of HA
oligomers, such as their TLR-4-mediated immunostimulatory
activity.
sulfonic anhydride),8 resulting in the formation of 1-thiodisac-
charides amenable for elongation at both the reducing end and
nonreducing end.9 We further elected to mask the reducing
glucuronide in our target HA oligomers as the 3-azido-1-
propanol glycoside, with the dual advantage of locking the
anomeric configuration and enabling functionalization of the
azide moiety for future conjugation studies.
Results and Discussion
The synthesis of functionalized monosaccharides 3, 4, 5, and
6 that we required for executing our strategy is summarized in
Scheme 1. Partially protected 1-phenylthioglucuronide 1, pre-
pared following our previously reported procedure,10 was
transformed into the corresponding 4-O-levulinoyl derivative
2. Glycosylation with 3-azidopropanol (Ph2SO, Tf2O) followed
by deblocking of the 4′-hydroxyl function gave reducing end
building block 3, whereas hydrolysis of the thioacetal in 2
provided 1-hydroxy donor 4.11 Partially protected glucosamine
derivative 5 was prepared as described by Blatter and
Jacquinet,12 and 3-O-levulinoylation gave donor thioglycoside 6.
The synthesis of fully protected HA trimer 8 is depicted in
Scheme 2 and commenced with the Ph2SO/Tf2O/TTBP-medi-
ated condensation of donor glycoside
4 and acceptor
glycoside 5. The resulting thiodisaccharide 7 was condensed
with acceptor glucuronide 3 under the same conditions to
provide trisaccharide 8.
At this stage, we investigated the efficiency in preparing
trisaccharide 8 using a one-pot procedure.13 Accordingly, the
reaction mixture containing disaccharide 7, formed after Tf2O/
Ph2SO-mediated condensation of 1-hydroxydonor 4 with thiogly-
coside 5, was cooled and preactivated with an additional 1 equiv
of Tf2O and TTBP (0.95 equiv with respect to Tf2O), followed
(8) (a) Code´e, J. D. C.; Stubba, B.; Schiattarella, M.; Overkleeft, H. S.;
van Boeckel, C. A. A.; van Boom, J. H.; van der Marel, G. A. J. Am. Chem.
Soc. 2005, 127, 3767-3773. (b) Garcia, B. A.; Poole, J. L.; Gin, D. Y. J.
Am. Chem. Soc. 1997, 119, 7597-7598.
Experimental Section
(9) (a) Code´e, J. D. C.; Litjens, R. E. J. N.; Den Heeten, R.; Overkleeft,
H. S.; van Boom, J. H.; van der Marel, G. A. Org. Lett. 2003, 5, 1519-
1522. (b) Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015-9020.
(10) van den Bos, L. J.; Code´e, J. D. C.; van Boom, J. H.; Overkleeft,
H. S.; van der Marel, G. A. Org. Lett. 2004, 6, 2165-2168.
(11) Dinkelaar, J.; Witte, M. D.; van den Bos, L. J.; Overkleeft, H. S.;
van der Marel, G. A. Carbohydr. Res. 2006, 341, 1723-1729.
(12) Blatter, G.; Jacquinet, J.-C. Carbohydr. Res. 1996, 288, 109-125.
(13) Code´e, J. D. C.; van den Bos, L. J.; Litjens, R. E. J. N.; Overkleeft,
H. S.; van Boom, J. H.; van der Marel, G. A. Org. Lett. 2003, 5, 1947-
1950.
Methyl (Phenyl 2,3-di-O-benzoyl-4-O-levulinoyl-1-thio-â-D-
glucopyranoside) Uronate (2). To a solution of 1 (3.81 g, 7.49
mmol) in pyridine (75 mL) was added a solution of Lev2O in
dioxane (0.5 M, 37.5 mL, 18.7 mmol). After 18 h the mixture was
diluted with EtOAc (200 mL) and washed with 1 M HCl (aq),
NaHCO3 (aq), and brine. The organic layer was dried over MgSO4
and concentrated in Vacuo. Purification by column chromatography
yielded 2 as a colorless oil (3.91 g, 86%). [R]D22: +63 (c ) 1,
5738 J. Org. Chem., Vol. 72, No. 15, 2007

Synthesis of Hyaluronic Acid Oligomers
SCHEME 2. Sequential (a) and One-Pot (b) Glycosylation Strategy
SCHEME 3. HA Tetramer and Pentamer Synthesis
1
CHCl3); IR (neat): 716, 1068, 1263, 1710, 2930 cm-1; H NMR
added, and the reaction mixture was allowed to warm to 0 °C. Dry
Et3N (1.39 mL, 10 mmol) was added, and the reaction was washed
with NaHCO3 (aq). The organic layer was dried over MgSO4 and
concentrated in Vacuo. This crude concentrate was then dissolved
in a mixture of pyridine (16 mL) and AcOH (4 mL), after which
hydrazine monohydrate (0.48 mL, 10 mmol) was added. The
mixture was stirred for 15 min, diluted with EtOAc (50 mL), and
washed with 1 M HCl (aq), NaHCO3 (aq), and brine. The organic
layer was dried over MgSO4 and concentrated in Vacuo. Purification
by column chromatography yielded 3 as a colorless oil (0.639 g,
64%). [R]D22: +56 (c ) 1, CHCl3); IR (neat): 709, 1067, 1252,
(400 MHz, CDCl3): δ ) 2.04 (s, 3H, CH3 Lev), 2.37-2.58 (m,
4H, 2 × CH2 Lev), 3.81 (s, 3H, CH3 COOMe), 4.23 (d, 1H, J )
10.0 Hz, H-5), 4.97 (d, 1H, J ) 10.0 Hz, H-1), 5.41 (m, 2H, H-2,
H-4), 5.71 (t, 1H, J ) 10.0 Hz, H-3),7.29-7.55 (m, 11H, CHarom),
7.87 (d, 2H, J ) 7.2 Hz, CH Bz), 7.93 (d, 2H, J ) 7.2 Hz, CH
Bz); 13C NMR (100 MHz, CDCl3) δ ) 27.7 (CH2 Lev), 29.5 (CH3
Lev), 37.6 (CH2 Lev), 53.0 (CH3 COOMe), 69.5, 70.0 (C-2, C-4),
73.5 (C-3), 76.4 (C-5), 86.6 (C-1), 128.4-128.7 (CHarom), 129.0
(Cq arom), 129.8-130.0 (CHarom), 131.3 (Cq arom), 133.4-133.5
(CHarom), 164.9, 165.6, 166.9, 171.1 (CdO Bz, COOMe, CdO
Lev), 205.5 (CdO Lev); HRMS: C32H30O10S + H+ requires
607.16324, found 607.16324.
3-Azidopropyl (Methyl (2,3-di-O-benzoyl-â-D-glucopyrano-
side) uronate) (3). A mixture of 2 (1.21 g, 2.00 mmol) and Ph2SO
(0.485 g, 2.40 mmol) were coevaporated with toluene two times
to remove traces of water, dissolved in DCM (40 mL), and further
dried by stirring over molecular sieves for 15 min. At -60 °C Tf2O
(0.40 mL, 2.4 mmol) was added. After 15 min a solution of
3-azidopropanol (0.608 g, 6.00 mmol) in DCM (15 mL) was slowly
1
1717, 2103, 2930 cm-1; H NMR (400 MHz, CDCl3): δ ) 1.78
(m, 2H, CH2 C3H6N3), 3.25 (m, 2H, CH2 C3H6N3), 3.37 (d, 1H, J
) 2.4 Hz, OH), 3.63 (m, 1H, CH2 C3H6N3), 3.87 (s, 3H, CH3
COOMe), 4.02 (m, 1H, CH2 C3H6N3), 4.10 (m, 1H, H-3), 4.10 (dt,
1H, J ) 2.4 Hz, 9.2 Hz, H-4), 4.75 (d, 1H, J ) 7.6 Hz, H-1), 5.43
(dd, 1H, J ) 8.0 Hz, 9.6 Hz, H-2), 5.54 (dd, 1H, J ) 9.2 Hz, 9.6
Hz, H-5), 7.39 (m, 4H, CH Bz), 7.51 (m, 2H, CH Bz), 7.97 (m,
4H, CH Bz); 13C NMR (100 MHz, CDCl3) δ ) 28.9 (CH2 C3H6N3),
47.8 (CH2 C3H6N3), 53.0 (CH3 COOMe), 66.9 (CH2 C3H6N3), 70.6
J. Org. Chem, Vol. 72, No. 15, 2007 5739

Dinkelaar et al.
(C-4), 71.2 (C-2), 74.6 (C-5), 74.9 (C-3), 101.4 (C-1), 128.4 (CH
Bz), 128.4 (Cq Bz), 128.9 (Cq Bz), 129.7 (CH Bz), 129.9 (CH Bz),
133.4 (CH Bz), 165.1, 166.6, 169.1 (CdO Bz, COOMe); HRMS:
C24H25N3O9 + Na+ requires 522.14830, found 522.14827.
1H, J ) 6.8 Hz, NH), 7.29-7.43 (m, 16H, CHarom), 7.83 (m, 4H,
CHarom); 13C NMR (100 MHz, CDCl3) δ ) 27.6 (CH2 Lev), 29.6
(CH3 Lev), 37.5 (CH2 Lev), 52.9 (CH3 COOMe), 57.4 (C-2), 68.6
(C-6), 69.3 (C-2′), 70.5 (C-5), 71.9 (C-4′), 72.0 (C-5′), 72.5 (C-
3′), 77.0 (C-3), 79.7 (C-4), 84.1 (C-1), 99.3 (C-1′), 101.5 (CHPh),
126.0 (CHarom), 128.4-128.7 (CHarom), 128.8 (Cq arom),129.2-129.9
(CHarom), 131.1 (Cq arom), 133.4-133.5 (CHarom), 136.9 (Cq arom),
161.7, 164.9, 165.5, 167.0, 171.1 (CdO TCA, CdO Bz, CdO
COOMe, CdO lev), 205.5 (CdO lev); HRMS: C47H44Cl3NO15S
Methyl (2,3-Di-O-benzoyl-4-O-levulinoyl-D-glucopyranose) Ur-
onate (4). To a vigorously stirred solution of 2 (0.30 g, 0.50 mmol)
in CH2Cl2 (5 mL) and H2O (0.5 mL) were added at 0 °C NIS (112
mg, 0.50 mmol) and TFA (39 µL, 0.50 mmol). After TLC analysis
showed complete consumption of starting material, the reaction was
quenched with Na2S2O3 (aq) and washed with NaHCO3 (aq). The
organic layer was dried over MgSO4 and concentrated in Vacuo.
Purification by column chromatography yielded 4 as a colorless
oil (0.21 g, 82%). Spectral data of the major anomer R. IR (neat):
+
+ NH4 requires 1017.18355, found 1017.18301.
3-Azidopropyl (Methyl (2,3-di-O-benzoyl-4-O-(4,6-O-ben-
zylidene-2-deoxy-2-trichloroacetamido-3-O-(methyl (2,3-di-O-
benzoyl-4-O-levulinoyl-â-D-glucopyranosyl) uronate)-â-d-glu-
copyranosyl)-â-D-glucopyranoside) Uronate (8). A mixture of
1-thio donor 7 (0.314 g, 0.314 mmol), Ph2SO (0.070 g, 0.345
mmol), and TTBP (0.078 g, 0.314 mmol) was coevaporated with
toluene two times to remove traces of water, dissolved in DCM (6
mL), and further dried by stirring over molecular sieves for 15 min.
At -60 °C Tf2O (55 µL, 0.329 mmol) was added, and after 15
min at -60 °C a solution of acceptor 3 (0.191 g, 0.377 mmol) in
DCM (3 mL) was slowly added and the reaction mixture was
allowed to warm to 0 °C. Dry Et3N (0.44 mL, 3.14 mmol) was
added, the reaction was washed with NaHCO3 (aq), and the organic
layer was dried over MgSO4 and concentrated in Vacuo. Purification
by column chromatography yielded 8 as a colorless oil (0.204 g,
1
711, 1264, 1722, 2343, 2361, 2927, 3440 cm-1; H NMR (400
MHz, CDCl3): δ ) 2.03 (s, 3H, CH3 Lev), 2.40 (m, 1H, CH2 Lev),
2.60 (m, 3H, CH2 Lev), 3.74 (s, 3H, CH3 COOMe), 4.75 (d, 1H,
J ) 10.0 Hz, H-5), 5.08 (d, 1H, J ) 4.4 Hz, OH), 5.24 (dd, 1H, J
) 3.2 Hz, 10 Hz, H-2), 5.43 (dd, 1H, J ) 10.0 Hz, 9.6 Hz, H-4),
5.78 (d, 1H, J ) 3.6 Hz, H-1), 6.06 (dd, 1H, J ) 10.0 Hz, 9.6 Hz,
H-3), 7.33 (m, 4H, CH Bz), 7.48 (m, 2H, CH Bz), 7.94 (m, 4H,
CH Bz); 13C NMR (100 MHz, CDCl3) δ ) 27.6 (CH2 Lev), 29.3
(CH3 Lev), 37.5 (CH2 Lev), 52.8 (CH3 COOMe), 67.9 (C-5), 69.5
(C-3, C-4), 71.6 (C-2), 90.2 (C-1), 128.2 (CHBz), 128.7 (CqBz),
129.6 (CH Bz), 133.3 (CH Bz), 165.6, 165.8, 168.6, 171.3 (CdO
Bz, CdO COOMe, CdO Lev), 206.2 (CdO Lev); HRMS:
C26H26O11 + H+ requires 515.15479, found 515.15500.
22
47%). [R]D +31 (c ) 1, CHCl3); IR (neat): 709, 1027, 1045,
1
1267, 1726, 2099, 2361, 2927, 3338 cm-1; H NMR (400 MHz,
Phenyl 4,6-O-Benzylidene-2-deoxy-3-O-levulinoyl-2-trichlo-
roacetamido-1-thio-â-D-glucopyranoside (6). To a solution of 5
(0.505 g, 1.00 mmol) in pyridine (10 mL) was added a solution of
Lev2O in dioxane (0.5 M, 5.0 mL, 2.5 mmol). After 18 h the mixture
was diluted with EtOAc (mL) and washed with 1 M HCl (aq),
NaHCO3 (aq), and brine. The organic layer was dried over MgSO4
and concentrated in Vacuo. Purification by column chromatography
yielded 6 as a off-white solid (0.523 g, 87%). [R]D22: -47 (c ) 1,
CDCl3): δ ) 1.75 (m, 2H, CH2 C3H6N3), 2.01 (s, 3H, CH3 Lev),
2.32 (m, 1H, CH2 Lev), 2.51 (m, 3H, CH2 Lev, H-6′), 3.26 (m,
2H, CH2 C3H6N3), 3.32 (dt, 1H, J ) 4.8 Hz, 9.6 Hz, H-5′), 3.37
(q, 1H, J ) 9.6 Hz, H-2′), 3.46 (t, 1H, J ) 9.0 Hz, H-4′), 3.59 (m,
1H, CH2 C3H6N3), 3.63 (s, 3H, CH3 COOMe), 3.69 (dd, 1H, J )
4.8 Hz, 10.8 Hz, H-6′), 3.79 (s, 3H, CH3 COOMe), 3.79 (d, 1H, J
) 9.6 Hz, H-5′′), 3.94 (m, 1H, CH2 C3H6N3), 4.04 (d, 1H, J ) 9.6
Hz, H-5), 4.34 (t, 1H, J ) 9.0 Hz, H-4), 4.40 (t, 1H, J ) 9.6 Hz,
H-3′), 4.70 (d, 1H, J ) 7.2 Hz, H-1′′), 4.93 (d, 1H, J ) 7.8 Hz,
H-1), 5.11 (d, 1H, J ) 8.4 Hz, H-1′), 5.14 (s, 1H, CHPh), 5.34 (m,
3H, H-2, H-2′′, H-3′′), 5.49 (t, 1H, J ) 9.6 Hz, H-4′′), 5.57 (t, 1H,
J ) 9.6 Hz, H-3), 6.74 (d, 1H, J ) 7.8 Hz, NH), 7.31-7.58 (m,
17H, CHarom), 7.81 (d, 2H, J ) 7.2 Hz, CH Bz), 7.85 (d, 2H, J )
7.2 Hz, CH Bz), 7.93 (d, 2H, J ) 7.2 Hz, CH Bz), 7.99 (d, 2H, J
) 7.2 Hz, CH Bz); 13C NMR (100 MHz, CDCl3) δ ) 27.6 (CH2
Lev), 28.9 (CH2 C3H6N3), 29.6 (CH3 Lev), 37.6 (CH2 Lev), 47.8
(CH2 C3H6N3), 52.8 (CH3 COOMe), 53.2 (CH3 COOMe), 58.4 (C-
2′), 65.9 (C-5′), 66.9 (CH2 C3H6N3), 67.7 (C-6′), 69.4 (C-3′′), 71.5,
71.9 (C-2, C-2′′), 72.1 (C-5′′), 72.4 (C-3), 72.6 (C-4′′), 74.0 (C-5),
75.8 (C-4), 76.4 (C-3′), 79.6 (C-4′), 98.5 (C-1′), 99.6 (C-1), 101.2
(CHPh), 101.4 (C-1′′), 126.1 (CHarom), 128.3-128.9 (CHarom),
128.9-129.2 (Cq arom), 129.8-130.0 (CHarom), 133.3-133.4 (CHarom),
136.9 (Cq arom), 161.4, 164.9, 165.2, 165.3, 165.6, 167.0, 168.3,
171.1 (CdO TCA, CdO Bz, CdO COOMe, CdO lev), 205.7 (Cd
O Lev); HRMS: C65H63Cl3N4O24 + H+ requires 1389.29706, found
1389.29504.
CHCl3); IR (neat): 750, 824, 1081, 1534, 1688, 2882, 3312 cm-1
;
1H NMR (400 MHz, CDCl3): δ ) 2.08 (s, 3H, CH3 Lev), 2.54
(m, 2H, CH2 Lev), 2.67 (m, 2H, CH2 Lev), 3.52 (dd, 1H, J ) 9.6
Hz, 4.8 Hz, H-5), 3.69 (m, 2H, m, H-4, H-6), 4.04 (dd, 1H, J )
10.0 Hz, 9.6 Hz, H-2), 4.11 (dd, 1H, J ) 10.0 Hz, 4.8 Hz, H-6),
4.92 (d, 1H, J ) 10.4 Hz, H-1), 5.50 (m, 2H, H-3, CHPh), 7.23 (d,
1H, J ) 9.6 Hz, NH), 7.24 (m, 6H, CHarom), 7.46 (m, 4H, CHarom);
13C NMR (100 MHz, CDCl3) δ ) 28.0 (CH2 Lev), 29.6 (CH3 Lev),
37.9 (CH2 Lev), 55.0 (C-2), 68.2 (C-6), 70.6 (C-5), 72.4 (C-3),
78.3 (C-4), 87.1 (C-1), 92.3 (CCl3), 101.1 (CHPh), 126.0-129.1
(CHarom), 132.0 (Cq SPh), 132.8 (CHarom), 136.8 (Cq CHPh), 161.8,
173.1 (CdO TCA, CdO Lev), 205.6 (CdO Lev); HRMS: C26H26-
Cl3NO7S + Na+ requires 624.03878, found 624.03870.
Phenyl (4,6-O-Benzylidene-2-deoxy-2-trichloroacetamido-3-
O-(methyl (2,3-di-O-benzoyl-4-O-levulinoyl-â-D-glucopyranosyl)
uronate)-1-thio-â-D-glucopyranoside (7). A mixture of 1-hydroxy
donor 4 (0.514 g, 1.00 mmol), Ph2SO (0.485 g, 2.40 mmol), and
TTBP (0.248 g, 1.00 mmol) was coevaporated with toluene two
times to remove traces of water, dissolved in DCM (20 mL), and
further dried by stirring over molecular sieves for 15 min. At -60
°C Tf2O (0.177 mL, 1.05 mmol) was added and the temperature
was raised to -40 °C. After 1 h. a solution of acceptor 5 (0.505 g,
1.00 mmol) in DCM (20 mL) was slowly added, and the reaction
mixture was allowed to warm to 0 °C. Dry Et3N (1.35 mL, 10
mmol) was added, the reaction was washed with NaHCO3 (aq),
and the organic layer was dried over MgSO4 and concentrated in
Vacuo. Purification by column chromatography yielded 7 as a white
solid (0.314 g, 56%). IR (neat): 709, 1090, 1271, 1718, 2360, 2930,
3-Azidopropyl (Methyl (2,3-di-O-benzoyl-4-O-(4,6-O-ben-
zylidene-2-deoxy-2-trichloroacetamido-3-O-(methyl (2,3-di-O-
benzoyl-â-D-glucopyranosyl) uronate)-â-D-glucopyranosyl)-â-D-
glucopyranoside) uronate) (9). HA-trimer (8) (204 mg, 0.147
mmol) was dissolved in a mixture of pyridine (2.35 mL) and AcOH
(0.58 mL), after which hydrazine monohydrate (0.036 mL, 0.735
mmol) was added. The mixture was stirred for 15 min and diluted
with EtOAc (20 mL) and washed with 1 M HCl (aq), NaHCO3
(aq), and brine. The organic layer was dried over MgSO4 and
concentrated in Vacuo. Purification by column chromatography
yielded 9 as a white solid (182 mg, 96%). IR (neat): 705, 1027,
1
3334 cm-1; H NMR (400 MHz, CDCl3): δ ) 2.01 (s, 3H, CH3
Lev), 2.34 (m, 1H, CH2 Lev), 2.50 (m, 3H, CH2 Lev), 3.45 (m,
1H, H-2), 3.62 (dt, 1H, J ) 4.8 Hz, 9.6 Hz, H-5), 3.66 (s, 3H, CH3
COOMe), 3.81 (m, 3H, H-4, H-5′, H-6), 4.35 (dd, 1H, J ) 5.2 Hz,
10.8 Hz, H-6), 4.69 (t, 1H, J ) 9.2 Hz, H-3), 5.02 (d, 1H, J ) 7.6
Hz, H-1′), 5.37 (m, 2H, H-2′, H-4′), 5.44 (d, 1H, J ) 10.4 Hz,
H-1), 5.51 (s, 1H, CHPh), 5.54 (t, 1H, J ) 9.6 Hz, H-3′), 6.98 (d,
1091, 1264, 1711, 1734, 2098, 2343, 2360, 2890, 3374, 3503 cm-1
;
1H NMR (400 MHz, CDCl3): δ ) 1.76 (m, 2H, CH2 C3H6N3),
2.56 (t, 1H, J ) 10.4 Hz, H-6′), 3.16 (d, 1H, J ) 3.2 Hz, OH),
3.28 (m, 2H, CH2 C3H6N3), 3.34 (dd, 1H, J ) 4.8 Hz, 9.6 Hz,
H-5′), 3.42 (m, 2H, H-4′ H-2′), 3.59 (m, 1H, CH2 C3H6N3), 3.71
5740 J. Org. Chem., Vol. 72, No. 15, 2007

Synthesis of Hyaluronic Acid Oligomers
(m, 2H, H-6′, H-5′′), 3.73 (s, 3H, CH3 COOMe), 3.79 (s, 3H, CH3
COOMe), 3.94 (m, 1H, CH2 C3H6N3), 4.05 (d, 1H, J ) 9.2 Hz,
H-5), 4.09 (dd, 1H, J ) 3.2 Hz, 9.2 Hz, H-4′′), 4.34 (t, 1H, J )
9.2 Hz, H-4), 4.38 (t, 1H, J ) 9.2 Hz, H-3′), 4.70 (d, 1H, J ) 7.2
Hz, H-1′′), 4.93 (d, 1H, J ) 7.2 Hz, H-1), 5.10 (d, 1H, J ) 8.4 Hz,
H-1′), 5.18 (s, 1H, CHPh), 5.34 (m, 3H, H-2, H-2′′, H-3′′), 5.58 (t,
1H, J ) 9.2 Hz, H-3), 6.72 (d, 1H, J ) 8.0 Hz, NH), 7.31-7.58
(m, 17H, CHarom), 7.88 (m, 4H, CH Bz), 7.93 (m, 2H, CH Bz),
7.99 (m, 2H, CH Bz); 13C NMR (100 MHz, CDCl3) δ ) 28.9 (CH2
C3H6N3), 47.8 (CH2 C3H6N3), 52.7 (CH3 COOMe), 53.2 (CH3
COOMe), 58.3 (C-2′), 65.9 (C-5′), 66.9 (CH2 C3H6N3), 67.7 (C-
6′), 70.2 (C-4′′), 71.5, 71.7 (C-2, C-2′′), 72.5 (C-3), 74.0 (C-5),
74.1 (C-5′′), 75.1 (C-3′′), 75.9 (C-4), 76.2 (C-3′), 79.5 (C-4′), 98.8
(C-1′), 99.5 (C-1′′), 101.2 (C-1), 101.4 (CHPh), 125.9 (CHarom),
128.3-128.4 (CHarom), 128.9-129.2 (Cq arom), 129.7-130.0 (CHarom),
133.3-133.4 (CHarom), 137.0 (Cq arom), 161.4, 165.0, 165.1, 165.2,
166.4, 168.4, 169.0 (CdO TCA, CdO Bz, CdO COOMe);
benzoyl-4-O-(4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-
3-O-(methyl (2,3-di-O-benzoyl-4-O-levulinoyl-â-D-glucopyranosyl)
uronate)-â-D-glucopyranosyl)-â-D-glucopyranosyl) uronate)-â-
D-glucopyranosyl)-â-D-glucopyranoside) uronate) (13). A mixture
of 1-thio donor 7 (0.164 g, 0.164 mmol), Ph2SO (0.036 g, 0.177
mmol), and TTBP (0.039 g, 0.157 mmol) was coevaporated with
toluene two times to remove traces of water, dissolved in DCM
(3.3 mL), and further dried by stirring over molecular sieves for
15 min. At -60 °C Tf2O (29 µL, 0.171 mmol) was added, and
after 15 min at -60 °C a solution of trisaccharide acceptor 9 (0.143
g, 0.110 mmol) in DCM (1.1 mL) was slowly added and the reaction
mixture was allowed to warm to -15 °C. Dry Et3N (0.25 mL, 1.6
mmol) was added, the reaction was washed with NaHCO3 (aq),
and the organic layer was dried over MgSO4 and concentrated in
Vacuo. Purification by column chromatography yielded 13 as a
colorless oil (0.116 g, 48%). IR (neat): 709, 1027, 1069, 1267,
1728, 2100, 2342, 2360, 2926 cm-1; 1H NMR (400 MHz, CDCl3):
δ ) 1H NMR (400 MHz, CDCl3): δ ) 1.75 (m, 2H, CH2 C3H6N3),
2.01 (s, 3H, CH3 Lev), 2.45 (t, 1H, J ) 6.8 Hz, H-6′ or H-6′′′),
2.45 (t, 1H, J ) 6.4 Hz, H-6′ or H-6′′′), 2.35 (m, 1H, CH2 Lev),
2.51 (m, 3H, CH2 Lev), 3.25 (m, 5H, CH2 C3H6N3, H-5′, H-5′′′,
H-2′′′), 3.39 (m, 3H, H-2′, H-4′, H-4′′′), 3.59 (m, 1H, CH2 C3H6N3),
3.62 (s, 3H, CH3 COOMe), 3.63 (s, 3H, CH3 COOMe), 3.67 (m,
2H, H-6′, H-6′′′), 3.78 (m, 2H, H-5, H-5′′′′), 3.80 (s, 3H, CH3
COOMe), 3.85 (m, 1H, CH2 C3H6N3), 4.05 (d, 1H, J ) 9.6 Hz,
H-5′′), 4.30 (m, 3H, H-3′′′, H-4, H-4′′), 4.43 (t, 1H, J ) 9.2 Hz,
H-3′), 4.71 (d, 1H, J ) 7.2 Hz, H-1), 4.90 (d, 1H, J ) 8.0 Hz,
H-1′′ or H-1′′′′), 4.92 (d, 1H, J ) 8.0 Hz, H-1′′ or H-1′′′′), 5.04 (d,
1H, J ) 8.0 Hz, H-1′), 5.08 (d, 1H, J ) 8.4 Hz, H-1′′′), 5.10 (s,
1H, CHPh), 5.17 (s, 1H, CHPh), 5.22 (dd, 1H, J ) 6.8 Hz, 9.6 Hz,
H-2′′′′), 5.34 (m, 3H, H-2, H-2′′, H-4′′′′), 5.39 (t, 1H, J ) 9.2 Hz,
H-3′′′′), 5.48 (t, 1H, J ) 9.2 Hz, H-3), 5.57 (t, 1H, J ) 9.2 Hz,
H-3′′), 6.66 (d, 1H, J ) 7.2 Hz, NH), 6.72 (d, 1H, J ) 8.0 Hz,
NH), 7.27-7.57 (m, 28H, CHarom), 7.78-7.98 (m, 12H, CH Bz);
13C NMR (100 MHz, CDCl3) δ ) 27.6 (CH2 Lev), 28.9 (CH2
C3H6N3), 29.6 (CH3 Lev), 37.5 (CH2 Lev), 47.8 (CH2 C3H6N3),
52.8 (CH3 COOMe), 52.9 (CH3 COOMe), 53.2 (CH3 COOMe).
58.1 (C-2′), 58.5 (C-2′′′), 65.8, 65.9 (C-5′, C-5′′′), 66.9 (CH2
C3H6N3), 67.6 (C-6′ and C-6′′′), 69.3, 71.4, 71.9, 72.1, 72.2, 72.4,
72.6 (C-2, C-2′′, C-2′′′′, C-3, C-3′′, C-3′′′′), 73.8, 74.0 (C-5, C-5′′,
C-5′′′′), 75.4, 76.0, 76.2 (C-3′, C-3′′′, C-4, C-4′′, C-4′′′′), 79.3, 79.5
(C-4′, C-4′′′), 92.3 (Cq TCA), 98.4 (C-1′′′), 99.0 (C-1′), 99.5
(C-1′′), 99.8 (C-1′′′′), 100.8 (CHPh), 101.1 (CHPh), 101.4
(C-1), 125.8-126.2 (CHarom), 128.3-128.4 (CHarom), 128.7-129.4
(Cq arom), 129.7-123.0 (CHarom), 133.3-133.3 (CHarom), 136.9
(Cq arom), 137.0 (Cq arom), 161.4, 161.4, 164.8-165.5, 166.9, 168.1,
168.5, 171.1 (CdO TCA, CdO Bz, CdO COOMe, CdO lev),
+
HRMS: C60H57Cl3N4O22 + NH4 requires 1308.28683, found
1308.28478.
3-Azidopropyl (Methyl (2,3-di-O-benzoyl-4-O-(4,6-O-ben-
zylidene-2-deoxy-2-trichloroacetamido-3-O-(methyl (2,3-di-O-
benzoyl-4-O-(4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-2-trichlo-
roacetamido-<bold>â-D-glucopyranosyl)-â-D-glucopyranosyl)
uronate)-â-d-glucopyranosyl)-â-D-glucopyranoside) uronate) (11).
A mixture of 1-thio donor 6 (0.081 g, 0.135 mmol), Ph2SO (0.030
g, 0.148 mmol), and TTBP (0.034 g, 0.135 mmol) was coevaporated
with toluene two times to remove traces of water, dissolved in DCM
(2.7 mL), and further dried by stirring over molecular sieves for
15 min. At -60 °C Tf2O (24 µL, 0.141 mmol) was added, and
after 15 min at -60 °C a solution of trisaccharide acceptor 9 (0.145
g, 0.112 mmol) in DCM (1.1 mL) was slowly added and the reaction
mixture was allowed to warm to -15 °C. Dry Et3N (0.2 mL, 1.3
mmol) was added, and the reaction was washed with NaHCO3 (aq).
The organic layer was dried over MgSO4 and concentrated in Vacuo.
Purification by column chromatography yielded 11 as a colorless
oil (0.124 g, 62%). IR (neat): 708, 1027, 1070, 1265, 1718, 2100,
1
2342, 2360, 2926 cm-1; H NMR (400 MHz, CDCl3): δ ) 1.75
(m, 2H, CH2 C3H6N3), 2.10 (s, 3H, CH3 Lev), 2.45 (t, 1H, J )
10.4 Hz, H-6′′′), 2.54 (m, 3H, CH2 Lev, H-6′), 2.66 (m, 2H, CH2
Lev), 3.23 (m, 2H, CH2 C3H6N3), 3.29 (m, 2H, H-5′, H-5′′′), 3.39
(m, 2H, H-4′, H-4′′′), 3.46 (m, 2H, H-2′, H-6′′′), 3.61 (m, 1H, CH2
C3H6N3), 3.65 (s, 3H, CH3 COOMe), 3.69 (dd, 1H, J ) 4.4 Hz,
10.4 Hz, H-6′), 3.81 (s, 3H, CH3 COOMe), 3.87 (m, 2H, H-2′′′,
H-5), 3.94 (m, 1H, CH2 C3H6N3), 4.05 (d, 1H, J ) 9.2 Hz, H-5′′),
4.16 (t, 1H, J ) 9.2 Hz, H-4), 4.32 (t, 1H, J ) 9.6 Hz, H-3′), 4.33
(t, 1H, J ) 9.6 Hz, H-4′′), 4.71 (d, 1H, J ) 7.2 Hz, H-1′′), 4.87 (d,
1H, J ) 8.4 Hz, H-1′′′), 4.96 (d, 1H, J ) 6.8 Hz, H-1), 5.06 (d,
1H, J ) 8.0 Hz, H-1′), 5.14 (s, 1H, CHPh), 5.18 (s, 1H, CHPh),
5.22 (t, 1H, J ) 10.0 Hz, H-3′′′), 5.26 (t, 1H, J ) 6.8 Hz, H-2),
5.57 (dd, 1H, J ) 7.6 Hz, 9.6 Hz, H-2′′), 5.48 (t, 1H, J ) 9.6 Hz,
H-3), 5.58 (t, 1H, J ) 9.6 Hz, H-3′′), 6.71 (d, 1H, J ) 8.0 Hz,
NH), 6.88 (d, 1H, J ) 9.2 Hz, NH), 7.31-7.58 (m, 22H, CHarom),
7.85 (d, 2H, J ) 7.6 Hz, CH Bz), 7.91 (m, 4H, CH Bz), 7.99 (d,
2H, J ) 7.2 Hz, CH Bz); 13C NMR (100 MHz, CDCl3) δ ) 28.0
(CH2 Lev), 28.8 (CH2 C3H6N3), 29.7 (CH3 Lev), 37.9 (CH2 Lev),
47.8 (CH2 C3H6N3), 52.8 (CH3 COOMe), 53.2 (CH3 COOMe), 56.1
(C-2′′′), 58.1 (C-2′), 65.9 (C-5′), 66.1 (C-5′′′), 66.9 (CH2 C3H6N3),
67.4 (C-6′′′), 68.0 (C-6′), 71.4 (C-2′′), 71.7 (C-3′′), 72.2 (C-3), 72.3
(C-2), 72.4 (C-3′′), 73.4 (C-5), 73.9 (C-5′′), 76.0 (C-3′), 76.1 (C-
4′′), 77.0 (C-4), 78.0 (C-4′′′), 79.1 (C-4′), 92.3 (Cq TCA), 99.0 (C-
1′), 99.6 (C-1), 100.8 (CHPh), 101.0 (CHPh), 101.0 (C-1′′′), 101.4
(C-1′′), 125.7-126.1 (CHarom), 128.2-128.4 (CHarom), 128.9-129.2
(Cq arom), 129.6-129.9 (CHarom), 133.2-133.4 (CHarom), 136.7
(Cq arom), 137.0 (Cq arom), 161.4, 161.6, 164.9-165.1, 168.5, 168.9,
172.3 (CdO TCA, CdO Bz, CdO COOMe, CdO lev), 205.8 (Cd
O Lev); HRMS: C80H77Cl6N5O29 + H+ requires 1782.29081, found
1782.29053.
+
205.6 (CdO Lev); HRMS: C101H95Cl6N5O37 + NH4 + Na+
requires 1110.20338, found 1110.20361.
3-Azidopropyl (4-O-(2-Deoxy-2-acetamido-3-O-(â-D-glucopy-
ranuronic acid)-â-D-glucopyranosyl)-â-D-glucopyranuronic Acid
(10). Trisaccharide 8 (44 mg, 0.032 mmol) was dissolved in MeOH
(5 mL), and a catalytic amount of p-toluenesulfonic acid was added.
The reaction mixture was stirred for 15 h, quenched with Et3N (0.1
mL), and concentrated in Vacuo. The remaining syrup was taken
up in a mixture of THF and H2O (6 mL, 1/1 v/v), and a 0.5 M
solution of KOH in H2O (0.64 mL, 0.32 mmol) was added stepwise
(1 equiv) over a period of 48 h. The reaction mixture was stirred
for 4 days after which it was quenched with Amberlite H+,
concentrated in Vacuo, and desalted by gel filtration. The resulting
sugar was then taken up in MeOH (5 mL) and Ac2O (0.25 mL).
After 2 h this mixture was coevaporated three times with MeOH
and toluene (1/1 v/v) and concentrated in Vacuo. Purification by
gel filtration and lyophilization yielded 10 as a white solid (12 mg,
58%). 1H NMR (600 MHz, D2O): δ ) 1.83 (m, 2H, CH2 C3H6N3),
1.97 (s, 3H, CH3 NHAc), 3.26 (m, 2H, H-2, H-2′′), 3.85 (t, 2H, J
) 7.2 Hz, CH2 C3H6N3), 3.43-3.44 (m, 2H), 3.47-3.54 (m, 2H),
3.63-3.74 (m, 7H), 3.79 (t, 1H, J ) 8.4 Hz, H-2′), 3.86 (d, 1H, J
) 10.8 Hz, H-6′), 3.91 (m, 1H, CH2 C3H6N3), 4.40 (m, 2H, H-1,
3-Azidopropyl (Methyl (2,3-di-O-benzoyl-4-O-(4,6-O-ben-
zylidene-2-deoxy-2-trichloroacetamido-3-O-(methyl (2,3-di-O-
J. Org. Chem, Vol. 72, No. 15, 2007 5741

Dinkelaar et al.
H-1′′), 4.51 (d, 1H, J ) 8.4 Hz, H-1′); 13C NMR (150 MHz, D2O)
δ ) 23.4 (CH3 NHAc), 29.2 (CH2 C3H6N3), 48.8 (CH2 C3H6N3),
55.2 (C-2′), 61.5 (C-6′), 68.5 (CH2 C3H6N3), 69.4, 72.7, 73.6, 73.7,
74.8, 76.2, 76.3, 76.9, 77.6, 81.1, 83.9 (C-2, C-2′′, C-3, C-3′, C-3′′,
C-4, C-4′, C-4′′, C-5, C-5′, C-5′′), 101.6 (C-1′), 103.4 (C-1′′), 104.0
(C-1), 175.2, 175.9, 176.5 (CdO COOH, CdO NHAc); HRMS:
C23H36N4O18 + H+ requires 657.20974, found 657.20997.
3-Azidopropyl (4-O-(2-Deoxy-2-acetamido-3-O-(4-O-(2-deoxy-
2-acetamido-3-O-(â-D-glucopyranonic acid)-â-D-glucopyrano-
syl)-â-D-glucopyranuronic acid)-â-D-glucopyranosyl)-â-D-glu-
copyranuronic Acid (14). Pentasaccharide 13 (53 mg, 0.024 mmol)
was dissolved in MeOH (5 mL), and a catalytic amount of
p-toluenesulfonic acid was added. The reaction mixture was stirred
for 15 h, quenched with Et3N (0.1 mL), and concentrated in Vacuo.
The remaining syrup was taken up in a mixture of THF and H2O
(8 mL, 1/1 v/v), and a 0.5 M solution of KOH in H2O (0.72 mL,
0.36 mmol) was added stepwise (1 equiv) over a period of 64 h.
The reaction mixture was stirred for 12 days after which it was
quenched with Amberlite H+, concentrated in Vacuo, and desalted
by gel filtration. The resulting sugar was then taken up in MeOH
(5 mL) and Ac2O (0.25 mL). After 2 h this mixture was
coevaporated three times with toluene and concentrated in Vacuo.
Purification by gel filtration and lyophilization yielded 14 as a white
solid (12 mg, 48%). 1H NMR (600 MHz, D2O): δ ) 1.84 (m, 2H,
CH2 C3H6N3), 1.96 (s, 3H, CH3 NHAc), 1.97 (s, 3H, CH3 NHAc),
3.25-3.30 (m, 2H, H-2, H-2′′, H-2′′′′), 3.85 (m, 2H, CH2 C3H6N3),
3.43-3.45 (m, 4H), 3.46-3.50 (m, 2H), 3.51-3.54 (m, 2H), 3.61-
3.73 (m, 10H), 3.78 (m, 2H, H-2′, H-2′′′), 3.85 (m, 2H, H-6′, H-6′′′),
3.91 (m, 1H, CH2 C3H6N3), 4.40 (m, 3H, H-1, H-1′′, H-1′′′′), 4.50
(m, 2H, H-1′, H-1′′′); 13C NMR (150 MHz, D2O) δ ) 23.4 (CH3
NHAc), 29.2 (CH2 C3H6N3), 48.8 (CH2 C3H6N3), 55.2 (C-2′, C-2′′′),
61.4 (C-6′, C-6′′′), 68.4 (CH2 C3H6N3), 69.4, 69.5, 72.7, 73.4, 73.7,
74.5, 74.8, 76.2, 76.3, 76.3, 76.7, 77.3, 77.7, 80.8, 81.1, 83.5, 84.0
(C-2, C-2′′, C-2′′′′, C-3, C-3′, C-3′′, C-3′′′, C-3′′′′, C-4, C-4′, C-4′′,
C-4′′′, C-4′′′′, C-5, C-5′, C-5′′, C-5′′′, C-5′′′′), 101.5, 101.6 (C-1′,
C-1′′′), 103.4, 104.0, 104.2 (C-1, C-1′′, C-1′′′′), 175.1, 175.9, 176.5
(CdO COOH, CdO NHAc); HRMS: C37H57N5O29 + H+ requires
1036.32120, found 1036.32094.
3-Azidopropyl (4-O-(2-Deoxy-2-acetamido-3-O-(4-O-(2-deoxy-
2-acetamido-â-D-glucopyranosy l)-â-D-glucopyranuronic acid)-
â-D-glucopyranosyl)-â-D-glucopyranuronic Acid (12). Tetrasac-
charide 11 (80 mg, 0.045 mmol) was dissolved in MeOH (5 mL),
and a catalytic amount of p-toluenesulfonic acid was added. The
reaction mixture was stirred for 15 h, quenched with Et3N (0.1 mL),
and concentrated in Vacuo. The remaining syrup was taken up in a
mixture of THF and H2O (9 mL, 1/1 v/v), and a 0.5 M solution of
KOH in H2O (1 mL, 0.5 mmol) was added stepwise (1 equiv) over
a period of 48 h. The reaction mixture was stirred for 7 days after
which it was quenched with Amberlite H+, concentrated in Vacuo,
and desalted by gel filtration. The resulting sugar was then taken
up in MeOH (9 mL) and Ac2O (0.25 mL). After 2 h this mixture
was coevaporated three times with toluene and concentrated in
Vacuo. Purification by gel filtration and lyophilization yielded 12
1
as a white solid (21 mg, 54%). H NMR (600 MHz, D2O): δ )
1.83 (m, 2H, CH2 C3H6N3), 1.96 (s, 3H, CH3 NHAc), 1.99 (s, 3H,
CH3 NHAc), 3.25-3.30 (m, 2H, H-2, H-2′′), 3.37-3.40 (m, 4H),
3.43-3.48 (m, 2H), 3.50-3.54 (m, 2H), 3.62-3.72 (m, 10H),
3.77-3.80 (m, 1H), 3.85-3.87 (m, 2H, H-6′, H-6′′′), 3.91 (m, 1H,
CH2 C3H6N3), 4.40 (m, 2H, H-1, H-1′′), 4.47 (d, 1H, J ) 8.4 Hz,
H-1′ or H-1′′′), 4.50 (d, 1H, J ) 8.4 Hz, H-1′ or H-1′′′); 13C NMR
(150 MHz, D2O) δ ) 23.3 (CH3 NHAc), 23.4 (CH3 NHAc), 29.2
(CH2 C3H6N3), 48.8 (CH2 C3H6N3), 55.2, 56.3, 61.4 (C-2′, C-2′′′,
C-6′, C-′′′), 68.5 (CH2 C3H6N3), 69.4, 70.6, 73.4, 73.7, 74.5, 74.7,
76.3, 76.8, 77.2, 77.6, 80.7, 81.1, 83.4 (C-2, C-2′′, C-3, C-3′, C-3′′,
C-3′′′, C-4, C-4′, C-4′′, C-4′′′, C-5, C-5′, C-5′′, C-5′′′), 101.6 (C-1′
and C-1′′′), 103.4 (C-1 or C-1′′), 104.1 (C-1 or C-1′′), 175.1, 175.2,
175.8, 175.9 (CdO COOH, CdO NHAc); HRMS: C31H49N5O23
+ H+ requires 860.28911, found 860.28932.
Supporting Information Available: General experimental and
NMR spectra of all new compounds. This information is available
free of charge via the Internet at http://pubs.acs.org.
JO070704S
5742 J. Org. Chem., Vol. 72, No. 15, 2007