Synthesis of hyaluronic acid oligomers using chemoselective and one-pot strategies

Article abstract of DOI:10.1021/jo9003713

(Chemical Equation Presented) An efficient synthetic strategy toward a hyaluronic acid (HA) tri-, penta-, and heptamer having a glucosamine-reducing end is reported. The synthesis is based on a glucuronate ester thioglycoside and a trifluoro-N-phenylimida

Full text of DOI:10.1021/jo9003713

Synthesis of Hyaluronic Acid Oligomers using Chemoselective and
One-Pot Strategies
Jasper Dinkelaar, Henrik Gold, Herman S. Overkleeft, Jeroen D. C. Code´e,* 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 February 19, 2009
An efficient synthetic strategy toward a hyaluronic acid (HA) tri-, penta-, and heptamer having a
glucosamine-reducing end is reported. The synthesis is based on a glucuronate ester thioglycoside and a
trifluoro-N-phenylimidate glucosamine building block. The HA-fragments are synthesized using an
S-phenyl GlcN-GluA building block through a combination of chemoselective and one-pot condensation
strategies.
SCHEME 1. Retrosynthesis of Hyaluronan
Introduction
Hyaluronan (HA, 1, Scheme 1) is a linear glycosaminoglycan
polymer featuring the ꢀ-1,3-linked 2-acetamido-2-deoxy-D-
glucose-ꢀ-(1,4)-D-glucuronic acid disaccharide1 as repeating
unit. HA is involved in a wide variety of biological processes,
such as cell-migration, proliferation, adhesion, recognition,2
tumor invasion3 and tumor inhibition.4 Recently, evidence has
accumulated that specific activities of HA are related to the
length of the carbohydrate chain. For instance, whereas high
molecular weight HA polymers are immunosuppressive,5 small
HA oligosaccharides induce complete and irreversible matura-
tion of human dendritic cells through the Toll-like receptor 4
(TLR-4) and thereby activate the innate immune system.6 In
addition, activation of macrophages by HA oligomers, generated
by degradation with different types of glycosidases showed
different levels of interleukin-12 production, pointing toward
the relevance of the nature of the monosaccharide at the reducing
end of the HA oligomer (being either N-acetyl glucosamine
(GlcNAc) or glucuronic acid (GluA)).7
Since the first synthesis of a HA-disaccharide by Jeanloz in
1964,8 several routes of synthesis have been disclosed for the
construction of HA-oligomers having either a GlcA or a GlcNAc
at the reducing end.9,10 For example, Blatter and Jacquinet
reported the synthesis of tetra-, hexa- and octameric HA-
fragments having a ꢀ-O-methyl glucuronic acid terminus.11
Huang and Huang developed an preactivation based iterative
one-pot strategy for the synthesis of HA-fragments and could
incorporate either a glucuronic acid or a glucosamine moiety
at the reducing end.12 We previously reported the synthesis of
(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.
(7) Stern, R.; Asari, A. A.; Sugahara, K. N. Eur. J. Cell. Biol. 2006, 85,
699–715, and references cited therein.
(5) McBride, W. H.; Bard, J. B. J. Exp. Med. 1997, 149, 507–515.
(6) 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.
(8) Fowers, H. M.; Jeanloz, R. W. Biochemistry 1964, 3, 123–125.
(9) For a review on the synthesis of glycosaminoglycans, including HA, see:
Yeung, B. K. S.; Chong, P. Y. C.; Peuntilo, P. A. Carbohydr. Res. 2002, 21,
779–865.
4208 J. Org. Chem. 2009, 74, 4208–4216
10.1021/jo9003713 CCC: $40.75 2009 American Chemical Society
Published on Web 04/29/2009

Synthesis of Hyaluronic Acid Oligomers
TABLE 1. Glycosylations of 8, 9, and 12 with Acceptor 13
entry
donor
equiv. donor
activator, conditions
yield
1
2
3
5
4
6
8, R ) SPh
1.2
1.2
1.2
1.2
1.2
1.5
Ph2SO/Tf2O, -60 to 0 °C
Ph2SO /Tf2O, -60 to 0 °C
BSP/Tf2O, -60 to 0 °C
TMSOTf, 0 °C
TfOH, 0 °C
TfOH, 0 °C
70%
63%
56%
78%
79%
90%
9, R ) OH
9, R ) OH
12, R ) OC(dNPh)CF3
12, R ) OC(dNPh)CF3
12, R ) OC(dNPh)CF3
SCHEME 2. Synthesis of the Monomeric Building Blocks 8, 9 and 12
a HA-pentamer having a glucuronic acid moiety at the reducing
end13 using the glucuronate (GlcA)-glucosamine (GlcN) thio-
disaccharide building block 2 (Scheme 1) in combination with
the diphenylsulfoxide (Ph2SO)/trifluoromethanesulfonic anhy-
dride (Tf2O) activating system.14,15 In this paper, we present
an alternative assembly strategy for the synthesis of the
complementary oligomers, having a glucosamine reducing end
and ranging in size from three to seven monomers. The HA-
fragments are synthesized using GlcN-GlcA building block 3
(Scheme 1) and combines chemoselective and one-pot conden-
sation strategies.
was employed.17 In order to circumvent these drawbacks, we
decided to use the more acid stable di-tert-butylsilylidene
(DTBS) group for the protection of the C4 and C6 hydroxyls
of the glucosamine moiety in 3. As a first research objective,
we investigated chemoselective and orthogonal glycosylation
strategies to synthesize key building block 3 using 1-thio
glucuronate ester 13 in combination with 1-thio, 1-OH, or
1-trifluoro imidate glucosamine donor 8, 9 or 12 respectively
(Table 1). The synthesis of the three glucosamine building
blocks started with the introduction of the trichloroacetyl group
on the amino function of glucosamine 4, using trichloroacetyl-
chloride and triethylamine in methanol (Scheme 2).18 The
resulting N-TCA glucosamine 5 was then transformed into 1-thio
glucosamine 6 in three steps as described by Blatter and
Jacquinet.11 Thus, glucosamine 5 was per-acetylated, after which
the anomeric thiophenol function was introduced, and the acetyl
groups were removed. Introduction of the di-tert-butylsilylene
function on 6 was followed by levulinoylation of the C3-OH
to give glucosamine donor 8. Hydrolysis of the thioacetal in 8
using NIS/TFA then provided 1-hydroxy donor 9.19 1-Triflu-
oroimidate 12 was conveniently prepared in a three step
sequence from 5. Thus, regioselective silylation of the C6-OH
and C4-OH in 5 using di-tert-butylsilyl bistriflate in DMF at
Results and Discussion
In our previous synthesis of HA-oligomers, we found that
the acid instability of the benzylidene acetal in 2 necessitated
careful tuning of the amount of base (tritert-butylpyrimidine,
TTBP16) used in the glycosylation reactions.13 Use of too little
base gave unwanted cleavage of the benzylidene group, whereas
orthoester/oxazoline formation was observed when excess base
(10) (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) Yeung, B. K. S.; Hill, D. C.; Janicka, M.; Peuntilo, P. A. Org. Lett. 2000, 2,
1279–1282. (c) Iyer, S. S.; Rele, S. M.; Baskaran, S.; Chaikof, E. L. Tetrahedron
2003, 59, 631–638. (d) Lu, X. A.; Chou, C. H.; Wang, C. C.; Hung, S. C. Synlett
2003, 9, 1364–1366. (e) Palmacci, E. R.; Seeberger, P. H. Tetrahedron 2004,
60, 7755–7766.
(11) Blatter, G.; Jacquinet, J.-C. Carbohydr. Res. 1996, 288, 109–125.
(12) Huang, L.; Huang, X. Chem.-Eur. J. 2007, 13, 529–540.
(13) Dinkelaar, J.; Code´e, J. D. C.; Van den Bos, L. J.; Overkleeft, H. S.;
Van der Marel, G. A. J. Org. Chem. 2007, 72, 5737–5742.
(14) (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.
(15) (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.
(16) Crich, D.; Smith, M.; Yao, Q.; Picione, J. Synthesis 2001, 323–326.
(17) When orthoester or oxazoline formation is not possible in a glycosylation
reaction, we generally employ 2.5-3.0 equiv of TTBP with respect to the amount
of Tf2O used. If orthoester or oxazoline formation can occur, the use of base is
preferably avoided. In our previous HA-syntheses,13 we found that the use of
0.95 equiv of TTBP relative to the amount of Tf2O employed, led to the most
productive condenstaion reactions.
(18) The moderate yield (46%) of the isolated 2-deoxy-2-trichloroacetamido-
D-glucopyranose is due to the difficult purification and isolation of the product.
Preparation of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-trichloroacetamido-D-glucopy-
ranose from glucosamine•HCl, following a two step procedure (TCA-introduction
and per-acetylation) without purification of the intermediate trichloroacetamido
intermediate 5 yielded 77% of the target compound.
J. Org. Chem. Vol. 74, No. 11, 2009 4209

Dinkelaar et al.
SCHEME 3. Assembly of the Protected HA-Oligomers
-30 °C quantitatively yielded 4,6-O-di-tert-butylsilylidene
glucosamine 10. This diol was regioselectively transformed into
the anomeric 1-(N-phenyl)trifluoro imidate 11 with the use of
N-phenyltrifluoroacetimidoyl chloride (ClC(dNPh)CF3) and
Cs2CO3.20-22 Levulinoylation of the remaining 3-OH furnished
the completely protected imidate donor 12 in 67% yield from
5. Glucuronate 13 was synthesized from ꢀ-S-phenyl glucopy-
ranose as we described previously. Briefly, benzylidination and
ensuing benzoylation of ꢀ-S-phenyl glucopyranose gave the fully
protected glucopyranoside, of which the C-4 and C-6 hydroxyls
were liberated by acidic hydrolysis of the benzylidene acetal.
Subsequent oxidation of the primary alcohol function and
formation of the methyl ester gave glucuronate 13.23
The 1-thio (8), 1-hydroxy (9) and imidate (12) donors were
then glycosidated with glucuronate 13 as summarized in Table
1.24 The condensation of 1-thioglucosamine 8 with 13 proceeded
rapidly using the Ph2SO/Tf2O activator system14 and yielded
disaccharide 3 in 70% (entry 1). No activation of the glucuronate
ester (13) or thiodisaccharide 3 was found during this glyco-
sylation indicating the reactivity difference of the donor (8) and
acceptor/product (13/3).25 The Ph2SO/Tf2O mediated dehydra-
tive condensation of 1-hydroxy glucosamine 9 and thioacceptor
13 proceeded slowly to give dimer 3 (63% yield). The
1-benzenesulfinyl piperidine (BSP)/Tf2O15b reagent combination
in this dehydrative condensation appeared less productive than
the Ph2SO/Tf2O promoter. Finally, imidate donor 12 reacted
smoothly with the acceptor using a catalytic amount of
trimethylsilyl triflate (TMSOTf) or triflic acid (TfOH) to yield
disaccharide 3 in 78 and 79% yield respectively (entries 4 and
5). Thus, all three donor-acceptor combinations provided dimer
3 in satisfactory yields, with imidate 12 performing best. Given
the fact that imidate 12 is synthesized in only 4 steps from
glucosamine, we tried to improve the yield of the imidate
coupling by increasing the amount of donor glycoside. When
1.5 equiv of 12 were used in the triflic acid mediated glycosyl-
ation of 13, dimer 3 was obtained in 90% yield.
With key dimer 3 in hand, we set out to assemble the target
HA-oligosaccharides as depicted in Scheme 3. First, the reducing
end glucosamine 15 was synthesized by coupling 1-thio
glucosamine 8 or imidate 12 with azidopropanol and subsequent
delevulinoylation. This reducing-end building block was con-
densed with dimer 3 using the Ph2SO/Tf2O activator system.
Although preactivation of the thiodisaccharide proceeded
smoothly, the ensuing reaction with acceptor 15 did not go to
completion and trisaccharide 16 was isolated in 46% yield. We
have observed complete activation and poor condensation yields
with uronate ester donors before,14a and have previously been
able to increase the coupling efficiency by changing from
Ph2SO/Tf2O to the related BSP/Tf2O reagent system. Also in
the present case, this change in activator system significantly
improved the outcome of the glycosylation and we were able
(19) Dinkelaar, J.; Witte, M. D.; Van den Bos, L. J.; Overkleeft, H. S.; Van
der Marel, G. A. Carbohydr. Res. 2006, 341, 1723–1729.
(20) Valerio, S.; Pastore, A.; Adinolfi, M.; Iadonisi, A. J. Org. Chem. 2008,
73, 4496–4503.
(21) The anomeric configuration of the N-phenyltrifluoroacetimidate glu-
cosamines 11 and 12 was assigned based on the chemical shift of the anomic
proton and anomeric carbon resonances (11: dH-1 ) 6.40 ppm, dC-1 ) 93.0 ppm;
12 dH-1 ) 6.43, dC-1 ) 92.6 ppm).
(22) Attempted selective levulinoyl protection of the C3-OH in diol 10 led
to an inseparable mixture of compounds.
(23) 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.
(24) To compare the silylidene functionalized building block 7 with its
benzylidene protected counterpart we previously described,13 two trisaccharides
were assembled having either a silylidene or a benzylidene protected glucosamine.
Glycosylations using the silylidene protected glycosides gave significant higher
yields than the corresponding benzylidene protected glycosides. See Supporting
Information for experimental details.
(25) Code´e, J. D. C.; Van den Bos, L. J.; Litjens, R. E. J. N.; Overkleeft,
H. S.; Van Boeckel, C. A. A.; Van Boom, J. H.; Van der Marel, G. A.
Tetrahedron 2004, 60, 1057–1064.
4210 J. Org. Chem. Vol. 74, No. 11, 2009

Synthesis of Hyaluronic Acid Oligomers
SCHEME 4. Deprotection of the HA Oligomers
to obtain HA trimer 16 in 75% yield. We also examined
N-iodosuccinimide (NIS)/TfOH as activator system, since this
should provide an opportunity for a one-pot synthesis of
trisaccharide 16, combining imidate chemistry with iodonium
ion activation of the thiodisaccharide. Under the agency of NIS/
TfOH, dimer 3 and glucosamine 15 were condensed to give
trisaccharide 16 in 75% yield. Next, a one-pot procedure was
investigated.26 Accordingly, imidate 12 and 1-thio glucuronate
13 were combined and treated with a catalytic amount of TfOH
to produce the 1-thio disaccharide. Subsequently, acceptor 15
and NIS were added to this mixture at 0 °C, leading to the
formation of trisaccharide 16, which was isolated in 72% yield.
Interestingly, the NMR spectrum of 16 revealed a rather small
homonuclear coupling constant (JH1′-H2′) for the anomeric proton
of the glucuronate moiety (H-1′) of 4.4 Hz. Upon deprotection
of the oligosaccharides (Vide infra) the coupling constant
changes to 8.4 Hz, indicative of the ꢀ-glucuronic acid linkage
formed. The small coupling constant for the glucuronate
anomeric proton indicates that the glucuronate ester takes up a
stable under acidic conditions. The synthesis of the oligomers
builds on the chemoselective condensation of glucosamine
N-phenyltrifluoroimidate 12 and S-phenyl glucuronate ester 13,
which are both accessible using short, high yielding synthetic
routes. Monomers 12 and 13 are condensed to give the key
1-thio disaccharide building block 3 or combined in a one-pot
glycosylation sequence with azidopropanol glucosamine 15 to
produce the reducing end trimer 16. For the synthesis of the
higher HA-oligomers, iodonium ion activation of dimer building
block 3 proved to be more effective than the sulfonium based
activator systems.28
4
flattened C1-chair conformation, when positioned in between
two 4,6-O-di-tert-butylsilylidene glucosamine residues.27
To elongate trisaccharide 16, the C3′′-OLev was deprotected
and the resulting alcohol 17 was condensed with dimer 3. To this
end, the 1-thiodisaccharide was activated by BSP/Tf2O and treated
with acceptor 17. We observed that, while the activated disaccha-
ride decomposed in time, little to no glycosylation took place, and
we recovered unreacted acceptor. We therefore switched to a NIS-
TfOH glycosylation protocol which does not require preactivation
of the donor glycoside at low temperature. In addition, iodonium
activated glycosylations are more easily executed at higher
concentration than preactivation based sulfonium promoted cou-
pling reactions. Thus, donor 3, acceptor 17 and NIS were dissolved
in dichloromethane (0.1 M in acceptor) and cooled to 0 °C before
addition of a catalytic amount of TfOH. This time, complete
consumption of trisaccharide 17 was observed and pentamer 18
was obtained in 98% yield. Ensuing delevulinoylation of 18 gave
alcohol 19 which was elongated in a subsequent NIS/TfOH
mediated glycosylation with building block 3. This reaction proved
to be difficult to monitor, because of the similar polarities of the
reaction partners and product as well as the rather viscous nature
of the reaction mixture. Nonetheless, heptamer 20 was easily
separated from the smaller products in the reaction mixture by size-
exclusion chromatography on Sephadex LH-20, and isolated in
61% yield.
Experimental Section
2-Deoxy-2-trichloroacetamido-D-glucopyranose (5). To a mix-
ture of D-glucosamine-HCl (53.9 g, 250 mmol) in MeOH (625 mL)
and Et3N (70 mL, 500 mmol) trichloroacetyl chloride (TCACl) (28
mL, 250 mmol) was added dropwise at 0 °C. After 5 days, the
mixture was filtered and concentrated in Vacuo. Purification by
column chromatography (EtOAc, MeOH) yielded 5 as an off-white
solid (37.8 g, 46%). Analytical data were identical to those described
in literature previously.29
Phenyl 4,6-O-di-tert-butylsilylidene-2-deoxy-2-trichloroacetamido-
ꢀ-thio-D-glucopyranoside (7). To a solution of phenyl 2-deoxy-2-
trichloroacetamido-ꢀ-thio-D-glucopyranoside (6) (6.95 g, 16.8
mmol) in DMF (80 mL) at -30 °C was added di-tert-butylsilylidene
bistriflate (5.42 mL, 16.8 mmol). The reaction was warmed to -10
°C in 1 h after which pyridine (4.0 mL, 50 mmol) was added and
subsequently, the reaction was diluted with Et2O and washed with
H2O. The organic layer was dried over MgSO4 and concentrated
in Vacuo. Purification by column chromatography (PE, EtOAc)
yielded 7 as a white amorphous solid (8.44 g, 90%). [R]D22 ) -18
(c ) 0.1, DCM); IR (neat): 818, 1063, 1528, 1687, 2359, 2887,
1
2931, 3335 cm-1; H NMR (400 MHz, CDCl3): δ ) 0.94 (s, 9H,
tBu), 1.04 (s, 9H, tBu), 2.99 (d, 1H, J ) 2.0 Hz, OH), 3.48 (dt,
1H, J ) 5.2 Hz, 9.6 Hz, H-5), 3.69-3.76 (m, 2H, H-2, H-4), 3.91
(t, 1H, J ) 10.0 Hz, H-6), 3.99 (dt, 1H, J ) 1.6 Hz, 8.4 Hz, H-3),
4.19 (dd, 1H, J ) 4.8 Hz, 5.2 Hz, H-6), 5.11 (d, 1H, J ) 10.4 Hz),
7.28-7.33 (m, 4H, NH, H Arom), 7.45-7.48 (m, 2H, H Arom);13C
NMR (100 MHz): δ ) 19.8 (Cq tBu), 22.5 (Cq tBu), 26.8 (CH3
tBu), 27.3 (CH3 tBu), 56.4 (C-2), 65.9 (C-6), 74.2 (C-5), 74.3 (C-
3), 77.3 (C-4), 86.1 (C-1), 92.3 (Cq CCl3), 128.2 (CH Arom), 128.9
(CH Arom), 132.0 (Cq Arom), 132.9 (CH Arom), 162.0 (CdO
TCA); HRMS: C22H32Cl3NO5SSi + H+ requires 556.0909, found
556.0907.
The synthesis of the HA-fragments was completed by global
deprotection of the oligomers 16, 18 and 20 as depicted in
Scheme 4. The silylidene groups were removed with Et3N/3HF,
and subsequent saponification of the ester and amide function-
alities using KOH in THF/H2O yielded the zwitterionic tri-,
penta-, and heptamer 21, 23, and 25. Finally, N-acetylation with
Ac2O in MeOH and subsequent basic treatment in H2O provided
the anionic HA-fragments 22, 24 and 26.
In conclusion, we have described the highly efficient synthesis
of a set of HA oligosaccharides combining chemoselective and
one-pot glycosylation strategies. The 4,6-silylidene function is
a valuable alternative to the benzylidene functionality we
previously employed in our HA syntheses, since the former is
Phenyl 4,6-O-di-tert-butylsilylidene-2-deoxy-4-O-levulinoyl-2-
trichloroacetamido-ꢀ-thio-D-glucopyranoside (8). To a solution of
7 (8.44 g, 15.2 mmol) in DCM (40 mL) at 0 °C was added levulinic
acid (LevOH) (3.87 g, 33.3 mmol), di-iso-propylcarbodiimine (DIC)
(2.58 mL, 16.7 mmol) and a catalytic amount of 4-dimethylami-
nopyridine (DMAP) were added. The mixture was stirred for four
hours and allowed to warm to RT. Filtration over Celite and
purification by column chromatography (PE, EtOAc) yielded 8 as
(26) 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.
(27) A similar coupling constant was observed for the glucuronate ester
anomeric protons in pentamer 18 and heptamer 20. The anomeric proton of the
glucuronate moiety in dimer 3 has a regular trans-diaxial coupling constant of
10.0 Hz.
(28) Crich, D.; Wu, B.; Jayalath, P. J. Org. Chem. 2007, 72, 6806–6815.
(29) Blatter, G.; Beau, J.-M.; Jacquinet, J.-C. Carbohydr. Res. 1994, 260,
189–202.
J. Org. Chem. Vol. 74, No. 11, 2009 4211

Dinkelaar et al.
a white amorphous solid (10.19 g, quant.). [R]D22 ) -26 (c ) 0.1,
Arom), 7.27 (m, 2H, H Arom);13C NMR (100 MHz): δ ) 19.9 (Cq
tBu), 22.7 (Cq tBu), 26.8 (CH3 tBu), 27.3 (CH3 tBu), 54.3 (C-2),
66.0 (C-6), 68.5 (C-5), 71.3 (C-3), 77.0 (C-4), 92.0 (Cq CCl3), 93.0
(C-1), 119.2 (CH Arom), 124.7 (CH Arom), 128.8 (CH Arom),
142.8 (Cq Arom), 162.2 (CdO TCA). HRMS: C24H32Cl3F3N2O6Si
+ H+ requires 635.1120, found 635.1120.
DCM); IR (neat): 825, 1070, 1166, 1525, 1701, 2341, 2360, 2860,
1
2933, 3315 cm-1; H NMR (400 MHz, CDCl3): δ ) 0.95 (s, 9H,
tBu), 1.04 (s, 9H, tBu), 2.15 (s, 3H, CH3 Lev), 2.57-2.61 (m, 2H,
CH2 Lev), 2.70-2.73 (m, 2H, CH2 Lev), 3.54 (dt, 1H, J ) 5.2 Hz,
10.0 Hz, H-5), 3.88-3.98 (m, 3H, H-2, H-4, H-6), 4.24 (dd, 1H, J
) 4.8 Hz, 5.2 Hz, H-6), 4.92 (d, 1H, J ) 10.4 Hz, H-1), 5.18 (dd,
1H, J ) 9.2 Hz, 9.2 Hz, H-3), 6.88 (d, 1H, J ) 9.2 Hz, NH),
7.31-7.34 (m, 3H, H Arom), 7.46-7.48 (m, 2H, H Arom);13C
NMR (100 MHz): δ )19.8 (Cq tBu), 22.6 (Cq tBu), 26.8 (CH3
tBu), 27.3 (CH3 tBu), 28.0 (CH2 Lev), 29.7 (CH3 Lev), 38.0 (CH2
Lev), 54.7 (C-2), 66.0 (C-6), 74.6, (C-5), 74.9 (C-4), 75.1 (C-3),
87.3 (C-1), 92.3 (Cq CCl3), 128.5 (CH Arom), 129.1 (CH Arom),
132.0 (Cq Arom), 133.0 (CH Arom), 161.7 (CdO TCA), 172.5
(CdO COO Lev), 205.8 (CdO CO Lev); HRMS: C27H38Cl3NO7SSi
+ H+ requires 654.1277, found 654.1278.
(N-Phenyl)-2,2,2-trifluoroacetimidate 4,6-O-di-tert-butylsilylidene-
2-deoxy-4-O-levulinoyl-2-trichloroacetamido-r/ꢀ-D-glucopyrano-
side (12). Imidate 11 (6.64 g, 10.0 mmol) was dissolved in DCM
(40 mL) and after cooling to 0 °C LevOH (3.28 g, 28.3 mmol),
DIC (2.2 mL, 14.2 mmol) and a catalytic amount of DMAP were
added. The mixture was stirred for four hours and allowed to warm
to RT. Filtration over Celite and purification by column chroma-
tography (PE, Et2O) yielded 12 as a colorless oil (R/ꢀ >10:1, 6.90 g,
94%).221H NMR (400 MHz, CDCl3): δ ) 0.99 (s, 9H, tBu), 1.07
(s, 9H, tBu), 2.13 (s, 3H, CH3 Lev), 2.62-2.65 (m, 2H, CH2 Lev),
2.70-2.75 (m, 2H, CH2 Lev), 3.88-3.99 (m, 2H, H-5, H-6), 3.98
(t, 1H, J ) 9.2 Hz, H-4), 4.14-4.19 (m, 1H, H-6), 4.26-4.28 (m,
1H, H-2), 5.27 (t, 1H, J ) 10.0 Hz, H-3), 6.43 (bs, 1H, H-1), 6.78
(d, 2H, J ) 8.0 Hz, H Arom), 7.11 (t, 1H, J ) 7.6 Hz, H Arom),
6.84 (d, 1H, J ) 7.6 Hz, NH), 7.27 (t, 2H, J ) 7.6 Hz, H Arom);13C
NMR (100 MHz): δ ) 19.7 (Cq tBu), 22.4 (Cq tBu), 26.6 (CH3
tBu), 27.1 (CH3 tBu), 27.7 (CH2 Lev), 29.4 (CH3 Lev), 37.7 (CH2
Lev), 53.5 (C-2), 65.9 (C-6), 68.8 (C-5), 71.8 (C-3), 73.8 (C-4),
91.6 (Cq CCl3), 92.6 (C-1), 119.4 (CH Arom), 124.6 (CH Arom),
129.0 (CH Arom), 142.5 (Cq Arom), 162.0 (CdO TCA), 173.5
(CdO COO Lev), 205.2 (CdO CO Lev). HRMS:
C29H38Cl3F3N2O8Si + Na+ requires 755.1307, found 755.1310.
Methyl (phenyl 2,3-Di-O-benzoyl-4-O-(4,6-O-di-tert-butylsilylidene-
2-deoxy-3-O-levulinoyl-2-trichloroacetamido-ꢀ-D-glucopyranoside)-
ꢀ-D-glucopyranosyl) uronate (3). Method A. A mixture of 1-thio
donor 8 (0.157 g, 0.24 mmol) and Ph2SO (0.057 g, 0.28 mmol)
was coevaporated with toluene two times to remove traces of water,
dissolved in DCM (5 mL) and stirred over activated 3 Å molsieves
for 30 min. At -60 °C, Tf2O (40 µL, 0.24 mmol) was added and
after 15 min at -60 °C, a solution of acceptor 13 (0.102 g, 0.2
mmol) in DCM (2 mL) was slowly added and the reaction mixture
was allowed to warm to 0 °C in 3 h. Dry Et3N (0.13 mL) was
added and the reaction mixture was diluted with DCM and washed
with NaHCO3 (aq). The water layer was extracted twice with DCM,
the collected organic layers were dried over MgSO4 and concen-
trated in Vacuo. Purification by column chromatography (PE,
EtOAc) yielded 3 as a white foam (0.147 g, 70%).
Method B. A mixture of 1-hydroxy donor 9 (0.135 g, 0.24 mmol)
and Ph2SO (0.097 g, 0.48 mmol) was coevaporated with toluene
two times to remove traces of water, dissolved in DCM (5.6 mL)
and stirred over activated 3 Å molsieves for 30 min. At -60 °C,
Tf2O (42 µL, 0.25 mmol) was added and the temperature was raised
to -40 °C. After 1 h, a solution of acceptor 13 (0.102 g, 0.2 mmol)
in DCM (2 mL) was slowly added and the reaction mixture was
allowed to warm to 0 °C in 2 h. Dry Et3N (0.13 mL) was added
and the reaction mixture was diluted with DCM and washed with
NaHCO3 (aq). The water layer was extracted twice with DCM, the
collected organic layers were dried over MgSO4 and concentrated in
Vacuo. Purification by column chromatography (PE, EtOAc) yielded
3 as a white foam (0.126 g, 63%).
Method C. A mixture of 1-hydroxy donor 9 (0.135 g, 0.24
mmol) and BSP (0.10 g, 0.48 mmol) was coevaporated with toluene
two times to remove traces of water, dissolved in DCM (5.6 mL)
and stirred over activated 3 Å molsieves for 30 min. At -60 °C,
Tf2O (42 µL, 0.25 mmol) was added and the temperature was raised
to -40 °C. After 1 h, a solution of acceptor 13 (0.102 g, 0.2 mmol)
in DCM (2 mL) was slowly added and the reaction mixture was
allowed to warm to 0 °C in 2 h. Dry Et3N (0.13 mL) was added
and the reaction mixture was diluted with DCM and washed with
NaHCO3 (aq). The water layer was extracted twice with DCM, the
collected organic layers were dried over MgSO4 and concentrated
in Vacuo. Purification by column chromatography (PE, EtOAc)
yielded 3 as a white foam (0.118 g, 56%).
4,6-O-Di-tert-butylsilylidene-2-deoxy-4-O-levulinoyl-2-trichloro-
acetamido-r/ꢀ-D-glucopyranose (9). To a solution of 8 (0.655 g,
1.00 mmol) in DCM (10 mL) and H2O (1 mL) at 0 °C was added
NIS (0.225 g, 1.00 mmol) and TFA (77 µL, 1.0 mmol). After 30
min a second equivalent of NIS (0.225 g, 1.00 mmol) was added
and the reaction was stirred for an additional 30 min. The reaction
was quenched by addition of Et3N and washed with Na2S2O3. The
organic layer was dried over MgSO4 and concentrated in Vacuo,
purification by column chromatography (PE, EtOAc) yielded 9 as
a colorless oil (0.838 g, 84%). IR (neat): 765, 817, 1064, 1092,
1533, 1701, 1747, 2337, 2931 cm-1; NMR assignment of major
1
isomer (R) H NMR (400 MHz, CDCl3): δ ) 0.98 (s, 9H, tBu),
1.05 (s, 9H, tBu), 2.17 (s, 3H, CH3 Lev), 2.60-2.64 (m, 2H, CH2
Lev), 2.72-2.75 (m, 2H, CH2 Lev), 3.34 (bs, 1H, OH), 3.89 (d,
1H, J ) 9.6 Hz, H-6), 3.93 (t, 1H, J ) 9.2 Hz, H-4), 4.06 (t, 1H,
J ) 4.8 Hz, H-5), 4.08-4.18 (m, 2H, H-2, H-6), 5.29 (dd, 1H, J )
9.2 Hz, 10.4 Hz, H-3), 5.32 (d, 1H, J ) 3.2 Hz, H-1), 7.06 (d, 1H,
J ) 8.8 Hz, NH);13C NMR (100 MHz): δ ) 19.9 (Cq tBu), 22.7
(Cq tBu), 26.8 (CH3 tBu), 27.3 (CH3 tBu), 28.1 (CH2 Lev), 29.8
(CH3 Lev), 38.0 (CH2 Lev), 54.0 (C-2), 66.4 (C-6), 66.8, (C-5),
72.5 (C-3), 74.9 (C-4), 87.3 (C-1), 91.3 (C-1), 162.1 (CdO TCA),
173.0 (CdO COO Lev), 205.9 (CdO CO Lev); HRMS:
C21H34Cl3NO8Si + H+ requires 562.1192, found 562.1192.
4,6-O-di-tert-butylsilylidene-2-deoxy-2-trichloroacetamido-r/ꢀ-
D-glucopyranose (10). To a solution of 5 (13.9 g, 43.0 mmol) in
DMF (215 mL) at -30 °C was added di-tert-butylsilylidene
bistriflate (13.6 mL, 42.0 mmol). The reaction was warmed to -10
°C in 1 h after which pyridine (10.9 mL, 129 mmol) was added
and subsequently the reaction was diluted with Et2O and washed
with H2O. The organic layer was dried over MgSO4 and concen-
trated in Vacuo to afford 10 as white amorphous solid (18.5 g, 95%).
IR (neat): 765, 817, 1064, 1092, 1533, 1683, 2337, 2931 cm-1
;
NMR assignment of major isomer (R), 1H NMR (400 MHz, CDCl3):
δ ) 0.99, (s, 9H, tBu), 1.06 (s, 9H, tBu), 3.30 (bs, 1H, OH),
3.74-3.82 (m, 1H), 3.87-3.94 (m, 2H), 3.96-4.13 (m, 3H, H-2,
H-6), 5.34 (d, 1H, J ) 3.2 Hz, H-1), 6.98 (d, 1H, J ) 8.4 Hz,
NH);13C NMR (100 MHz): δ ) 19.7 (Cq tBu), 22.7 (Cq tBu), 26.9
(CH3 tBu), 27.4 (CH3 tBu), 54.4 (C-2), 66.3 (C-6), 66.4 (C-5), 71.9
(C-3), 77.7 (C-4), 91.7 (C-1), 162.3 (CdO TCA). HRMS:
C16H28Cl3NO6Si + H+ requires 464.0824, found 464.0823.
(N-Phenyl)-2,2,2-trifluoroacetimidate 4,6-O-di-tert-butylsilylidene-
2-deoxy-2-trichloroacetamido-r/ꢀ-D-glucopyranoside (11). To a
solution of 10 (10.4 g, 22.4 mmol) in acetone (80 mL) at 0 °C was
added Cs2CO3 (8.0 g, 24.6 mmol) and ClC()NPh)CF3 (6.8 mL,
44.8 mmol). The reaction was warmed to RT, when TLC analysis
showed complete consumption of starting material the mixture was
filtered over Celite and concentrated in Vacuo. Purification by
column chromatography (PE, EtOAc) yielded 11 as a colorless oil
(R/ꢀ >10:1, 10.1 g, 71%).221H NMR (400 MHz, CDCl3): δ ) 1.01
(s, 9H, tBu), 1.07 (s, 9H, tBu), 2.84 (bs, 1H, OH), 3.84-3.93 (m,
3H, H-4, H-5, H-6), 3.98 (t, 1H, J ) 8.8 Hz, H-3), 4.16-4.20 (m,
2H, H-2, H-6), 6.40 (bs, 1H, H-1), 6.78 (d, 2H, J ) 7.6 Hz, H
Arom), 6.84 (d, 1H, J ) 7.2 Hz, NH), 7.11 (t, 1H, J ) 7.2 Hz, H
4212 J. Org. Chem. Vol. 74, No. 11, 2009

Synthesis of Hyaluronic Acid Oligomers
Method D. Imidate donor 12 (0.176 g, 0.24 mmol) and acceptor
13 (0.102 g, 0.20 mmol) in DCM (4 mL) were stirred over activated
3 Å molsieves for 30 min. The mixture was cooled to 0 °C before
a catalytic amount of TfOH (1 µL, 0.01 mmol) was added. The
mixture was allowed to warm to RT. After TLC analysis showed
complete consumption of starting material (1 h), the reaction was
quenched with Et3N. The reaction mixture was diluted with DCM
and washed with NaHCO3 (aq). The water layer was extracted twice
with DCM, the collected organic layers were dried over MgSO4
C3H6N3), 29.4 (CH3 Lev), 37.7 (CH2 Lev), 47.5 (CH2 C3H6N3),
55.4 (C-2), 65.8 (C-6), 66.1 (CH2 C3H6N3), 70.3, (C-5), 73.5 (C-
3), 74.7 (C-4), 92.2 (Cq CCl3), 100.7 (C-1), 162.0 (CdO TCA),
171.8 (CdO COO Lev), 205.8 (CdO CO Lev); HRMS:
C24H39Cl3N4O8Si + H+ requires 645.1676, found 645.1677.
3-Azidopropyl (4,6-O-di-tert-butylsilylidene-2-deoxy-2-trichlo-
roacetamido-ꢀ-D-glucopyranoside (15). Glucosamine 14 (0.579 g,
0.896 mmol) was dissolved in a mixture of pyridine (4 mL) and AcOH
(1 mL), after which hydrazine monohydrate (0.22 mL, 4.5 mmol) was
added. The mixture was stirred for 15 min and diluted with EtOAc
(20 mL), 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 (PE, EtOAc) yielded 15 as a
white amorphous solid (0.765 g, 85%).; IR (neat): 826, 1074, 1533,
1697, 2098, 2341, 2360, 2860, 2933, 3315 cm-1; 1H NMR (400 MHz,
CDCl3): δ ) 0.99 (s, 9H, tBu), 1.06 (s, 9H, tBu), 1.79-1.86 (m, 2H,
CH2 C3H6N3), 3.02 (s, 1H, OH), 3.37 (t, 2H, J ) 6.8 Hz, CH2 C3H6N3),
3.44-3.60 (m, 3H, H-2, H-5, CH2 C3H6N3), 3.70 (t, 1H, J ) 9.2 Hz,
H-4), 3.89-3.94 (m, 2H, H-6, CH2 C3H6N3), 4.02 (t, 1H, J ) 9.6 Hz,
H-3), 4.19 (dd, 1H, J ) 4.8 Hz, 5.2 Hz, H-6), 4.88 (d, 1H, J ) 8.4
Hz, H-1), 7.11 (d, 1H, J ) 6.7 Hz, NH);13C NMR (100 MHz): δ )
19.8 (Cq tBu), 22.6 (Cq tBu), 26.8 (CH3 tBu), 27.3 (CH3 tBu), 28.9
(CH2 C3H6N3), 47.9 (CH2 C3H6N3), 58.1 (C-2), 65.9 (C-6), 66.5 (CH2
C3H6N3), 70.2, (C-5), 72.6 (C-3), 77.7 (C-4), 92.4 (Cq CCl3), 99.8 (C-
1), 162.1 (CdO TCA); HRMS: C19H33Cl3N4O6Si + H+ requires
547.1308, found 547.1306.
3-Azidopropyl (4,6-O-di-tert-butylsilylidene-2-deoxy-2-trichlo-
roacetamido-3-O-(methyl (2,3-di-O-benzoyl-4-O-(4,6-O-di-tert-bu-
tylsilylidene-2-deoxy-3-O-levulinoyl-2-trichloroacetamido-ꢀ-D-glu-
copyranosyl)-ꢀ-D-glucopyranosyl) uronate)-ꢀ-D-glucopyranoside
(16). Method A. Thio dimer 3 (0.211 g, 0.2 mmol) and Ph2SO (0.045
g, 0.22 mmol) were coevaporated with toluene two times to remove
traces of water, dissolved in DCM (4 mL) and stirred over activated
3 Å molsieves for 30 min. At -60 °C, Tf2O (37 µL, 0.22 mmol) was
added and after 15 min at -60 °C a solution of acceptor 15 (0.132 g,
0.24 mmol) in DCM (2.4 mL) was slowly added and the reaction
mixture was allowed to warm to 0 °C in 3 h. Dry Et3N was added
and the reaction was washed with NaHCO3 (aq), the organic layer was
dried over MgSO4 and concentrated in Vacuo. Purification by size
exclusion and column chromatography (PE, EtOAc) yielded 16 as a
off white foam (0.137 g, 46%).
Method B. Thio dimer 3 (0.90 g, 0.86 mmol) and BSP (0.198 g,
0.946 mmol) were coevaporated with toluene two times to remove
traces of water, dissolved in DCM (17 mL) and stirred over activated
3 Å molsieves for 30 min. At -60 °C, Tf2O (0.152 mL, 0.903 mmol)
was added and after 15 min at -60 °C a solution of acceptor 15 (0.564
g, 1.03 mmol) in DCM (10 mL) was slowly added and the reaction
mixture was allowed to warm to 0 °C in 3 h. Dry Et3N (0.57 mL) was
added and the reaction was washed with NaHCO3 (aq), the organic layer
was dried over MgSO4 and concentrated in Vacuo. Purification by size
exclusion and column chromatography (PE, EtOAc) yielded 16 as a
off white foam (0.956 g, 75%).
Method C. Thio dimer 3 (0.211 g, 0.2 mmol) and acceptor 15
(0.132 g, 0.24 mmol) were coevaporated with toluene two times
to remove traces of water and dissolved in DCM (4 mL). NIS (0.054
g, 0.24 mmol) was added and the mixture was stirred over activated
3 Å molsieves for 30 min. The mixture was cooled to 0 °C before
a catalytic amount of TfOH (1 µL, 0.01 mmol) was added. After
TLC analysis showed complete consumption of thio dimer (1.5 h)
the reaction was quenched with Et3N. The reaction mixture was
and concentrated in Vacuo. Purification by column chromatography
22
(PE, EtOAc) yielded 3 as a white foam (0.164 g, 78%). [R]D
)
-18 (c ) 0.1, DCM); IR (neat): 826, 1074, 1533, 1697, 2098, 2341,
2361, 2860, 2933, 3315 cm-1; H NMR (400 MHz, CDCl3): δ )
1
0.878 (s, 18H, 2 x tBu), 2.14 (s, 3H, CH3 Lev), 2.52-2.59 (m, 3H,
H-6′, CH2 Lev), 2.64-2.70 (m, 2H, CH2 Lev), 3.24 (dt, 1H, J )
4.8 Hz, 10.0 Hz, H-5′), 3.45 (dd, 1H, J ) 4.8 Hz, 5.6 Hz, H-6′),
3.55 (t, 1H, J ) 9.2 Hz, H-4′), 3.80-3.86 (m, 4H, H-2′, CH3
COOMe), 4.14 (d, 1H, J ) 10.0 Hz, H-5), 4.24 (t, 1H, J ) 9.6 Hz,
H-4), 4.92 (d, 1H, J ) 8.4 Hz, H-1′), 4.99 (d, 1H, J ) 10.0 Hz,
H-1), 5.02 (t, 1H, J ) 10.4 Hz, H-3′), 5.38 (t, 1H, J ) 10.0 Hz,
H-2), 5.65 (t, 1H, J ) 9.2 Hz, H-3), 6.84 (d, 1H, J ) 9.2 Hz, NH),
7.28-7.53 (m, 11H, H Arom), 7.90-7.94 (m, 4H, H Arom);13C
NMR (100 MHz): δ )19.6 (Cq tBu), 22.3 (Cq tBu), 26.6 (CH3
tBu), 27.2 (CH3 tBu), 27.9 (CH2 Lev), 29.6 (CH3 Lev), 37.9 (CH2
Lev), 53.1 (CH3 COOMe), 55.6 (C-2′), 64.7 (C-6′), 69.5 (C-2),
70.4, (C-5′), 73.6 (C-3), 74.1 (C-3′), 74.3 (C-4′), 76.3 (C-4), 76.9
(C-5), 86.7 (C-1), 92.3 (Cq CCl3), 100.3 (C-1′), 128.3-129.6 (CH
Arom), 129.8 (Cq Arom), 131.5 (Cq Arom), 132.6-133.3 (CH
Arom), 161.5 (CdO TCA), 164.9 (CdO Bz), 165.0 (CdO Bz),
168.4 (CdO COOMe) 172.5 (CdO COO Lev), 205.8 (CdO CO
Lev); HRMS: C48H56Cl3NO15SSi + Na+ requires 1074.2098, found
1074.2112.
3-Azidopropyl (4,6-O-di-tert-butylsilylidene-2-deoxy-3-O-levuli-
noyl-2-trichloroacetamido-ꢀ-D-glucopyranoside (14). Method A.
A mixture of thio donor 8 (0.655 g, 1.0 mmol) and Ph2SO (0.233
g, 1.1 mmol) was coevaporated with toluene two times to remove
traces of water, dissolved in DCM (20 mL) and stirred over
activated 3 Å molsieves for 30 min. The mixture was cooled to
-78 °C before Tf2O (0.176 µL, 1.05 mmol) was added. The mixture
was stirred for 10 min at -78 °C followed by addition of
3-azidopropanol (0.303 g, 3.0 mmol) in DCM (6 mL). The reaction
mixture was allowed to warm to 0 °C in 4 h and Et3N (0.15 mL)
was added. The reaction mixture was diluted with DCM and washed
with NaHCO3 (aq). The water layer was extracted twice with DCM
after which the collected organic layers were dried over MgSO4
and concentrated in Vacuo. Purification by column chromatography
(PE, EtOAc) yielded 14 as a white amorphous solid (0.606 g, 94%).
Method B. Imidate donor 12 (0.176 g, 0.24 mmol) and
3-azidopropanol (0.073 g, 0.72 mmol) in DCM (4.8 mL) were
stirred over activated 3 Å molsieves for 30 min. The mixture was
cooled to 0 °C before a catalytic amount of TfOH (1 µL, 0.01 mmol)
was added, then the mixture was allowed to warm to RT. After
TLC analysis showed complete consumption of starting material
(1 h) the reaction was quenched with Et3N. The reaction mixture
was diluted with DCM and washed with NaHCO3 (aq). The water
layer was extracted twice with DCM, the collected organic layers
were dried over MgSO4 and concentrated in Vacuo. Purification
by column chromatography (PE, EtOAc) yielded 14 as a white
22
amorphous solid (0.143 g, 99%). [R]D ) -30 (c ) 0.1, DCM);
IR (neat): 827, 1078, 1558, 1699, 1716, 2098, 2341, 2361, 2860,
1
2933 cm-1; H NMR (400 MHz, CDCl3): δ ) 0.96 (s, 9H, tBu),
diluted with DCM and washed with Na2S2O3 (aq) and NaHCO3 (aq).
1.05 (s, 9H, tBu), 1.76-1.84 (m, 2H, CH2 C3H6N3), 2.15 (s, 3H,
CH3 Lev), 2.58-2.61 (m, 2H, CH2 Lev), 2.71-2.75 (m, 2H, CH2
Lev), 3.36 (t, 2H, J ) 6.8 Hz, CH2 C3H6N3), 3.48-3.60 (m, 2H,
H-5, CH2 C3H6N3), 3.89-4.02 (m, 4H, H-2, H-4, H-6, CH2
C3H6N3), 4.20 (dd, 1H, J ) 4.8 Hz, 5.2 Hz, H-6), 4.80 (d, 1H, J )
8.4 Hz, H-1), 5.28 (t, 1H, J ) 10.0 Hz, H-3), 7.72 (d, 1H, J ) 8.8
Hz, NH);13C NMR (100 MHz): δ )19.5 (Cq tBu), 22.3 (Cq tBu),
26.5 (CH3 tBu), 27.0 (CH3 tBu), 27.8 (CH2 Lev), 28.6 (CH2
The water layers were extracted twice with DCM and the collected
organic layers were dried over MgSO4 and concentrated in Vacuo.
Purification by size exclusion and column chromatography (PE,
EtOAc) yielded 17 as an off white foam (0.221 g, 75%).
Method D (1-Pot Procedure). Imidate donor 12 (1.36 g, 1.80
mmol) and acceptor 13 (0.610 g, 1.20 mmol) in DCM (18 mL)
were stirred over activated 3 Å molsieves for 30 min. The mixture
was cooled to 0 °C before a catalytic amount of TfOH (8 µL, 0.09
J. Org. Chem. Vol. 74, No. 11, 2009 4213

Dinkelaar et al.
mmol) was added, then the mixture was allowed to warm to RT
over approximately 1 h. After TLC analysis showed complete
consumption of thio acceptor (1 h), the mixture was cooled to 0
°C. Then, a mixture of glucosamine acceptor 15 (0.99 g, 1.60 mmol)
and NIS (0.32 g, 1.44 mmol) (dried over activated molecular sieves)
in DCM (18 mL) was added. After 2 h at 0 °C, TLC analysis
showed complete consumption of thio dimer and the reaction was
quenched with Et3N. The reaction mixture was diluted with DCM
and washed with NaHCO3 (aq). The water layer was extracted twice
with DCM, the collected organic layers were dried over MgSO4
and concentrated in Vacuo. Purification by size exclusion and
column chromatography (PE, EtOAc) yielded 16 as an off white
(C-2′′), 57.9 (C-2), 65.0 (C-6′′), 66.0 (C-6), 66.5 (CH2 C3H6N3),
70.1 (C-5′′), 70.3 (C-5), 71.5 (C-3′), 73.9 (C-2′), 74.1 (C-5′), 74.4
(C-4′′), 76.0 (C-4), 76.2 (C-4′), 77.3 (C-3′′), 77.9 (C-3), 92.5 (Cq
CCl3), 92.6 (Cq CCl3), 99.8 (C-1′), 99.9 (C-1′′), 100.0 (C-1),
128.3-128.8 (CH Arom, Cq Arom), 129.6-129.8 (CH Arom, Cq
Arom), 133.0-133.4 (CH Arom), 161.8 (CdO TCA), 162.2 (CdO
TCA), 165.2 (CdO Bz), 165.7 (CdO Bz), 170.2 (CdO COOMe);
HRMS: C56H77Cl6N5O19Si2 + H+ requires 1390.2955, found
1390.2975.
3-Azidopropyl (4,6-O-di-tert-butylsilylidene-2-deoxy-2-trichlo-
roacetamido-3-O-(methyl (2,3-di-O-benzoyl-4-O-(4,6-O-di-tert-bu-
tylsilylidene-2-deoxy-2-trichloroacetamido-3-O-(methyl (2,3-di-O-
benzoyl-4-O-(4,6-O-di-tert-butylsilylidene-2-deoxy-3-O-levulinoyl-
2-trichloroacetamido-ꢀ-D-glucopyranosyl)-ꢀ-D-glucopyranosyl) uro-
nate)-ꢀ-D-glucopyranosyl)-ꢀ-D-glucopyranosyl) uronate)-ꢀ-D-glu-
copyranoside (18). Thio dimer 3 (1.132 g, 1.074 mmol) and trimer
acceptor 17 (0.998 g, 0.716 mmol) were coevaporated with toluene
two times to remove traces of water then dissolved in DCM (10
mL). NIS (0.242 g, 1.074 mmol) was added and the mixture was
stirred over activated 3 Å molsieves for 30 min. The mixture was
cooled to 0 °C before a catalytic amount of TfOH (4 µL, 0.05 mmol)
was added, then the mixture was allowed to warm to RT. After
TLC analysis showed complete consumption of starting material
(2 h) the reaction was quenched with Et3N. The reaction mixture
was diluted with DCM and washed with NaHCO3 (aq). The water
layer was extracted twice with DCM and the collected organic layers
were dried over MgSO4 and concentrated in Vacuo. Purification
by size exclusion and column chromatography (PE, EtOAc) yielded
18 as a white foam (1.64 g, 98%). IR (neat): 825, 1069, 1527, 1724,
2099, 2860, 2933 cm-1; 1H NMR (400 MHz, CDCl3): δ ) 0.77
(s, 9H, tBu), 0.85 (s, 9H, tBu), 0.89 (s, 27H, tBu), 1.03 (s, 9H,
tBu), 1.78-1.80 (m, 2H, CH2 C3H6N3), 2.13 (s, 3H, CH3 Lev),
2.54-2.57 (m, 2H, CH2 Lev), 2.67-2.71 (m, 4H, H-6′′′′, H-6′′),
3.19-3.21 (m, 1H), 3.29-3.34 (m, 3H), 3.38-3.44 (m, 2H),
3.45-3.63 (m, 9H), 3.81 (s, 3H, CH3 COOMe), 3.83 (s, 3H, CH3
COOMe), 3.86-3.96 (m, 5H), 4.06-4.19 (m, 5H), 4.26-4.37 (m,
3H), 4.82 (d, 1H, J ) 8.0 Hz), 4.96-5.04 (m, 3H), 5.09-5.12 (m,
1H), 5.22-5.25 (m, 1H), 5.31-5.38 (m, 2H), 5.54-5.59 (m, 2H),
6.87-6.95 (m, 3H, NH), 7.35-7.42 (m, 8H, H Arom), 7.48-7.55
(m, 4H, H Arom), 7.88-7.92 (m, 8H, H Arom);13C NMR (100
MHz): δ ) 19.5 (Cq tBu), 19.7 (Cq tBu), 22.4 (Cq tBu), 22.6 (Cq
tBu), 26.6-26.7 (CH3 tBu), 27.1-27.2 (CH3 tBu), 28.0 (CH2 Lev),
28.9 (CH2 C3H6N3), 29.6 (CH3 Lev), 38.1 (CH2 Lev), 47.9 (CH2
C3H6N3), 52.8 (CH3 COOMe), 53.0 (CH3 COOMe), 55.6, 58.2,
58.7 (C-2, C-2′′, C-2′′′′), 65.1, 65.2, 66.1, (C-6, C-6′′, C-6′′′′), 66.6
(CH2 C3H6N3), 69.9, 70.4, 70.6, 71.3, 71.6, 73.8, 74.4, 74.4, 74.7,
74.8, 75.8, 75.9, 77.9, 78.3, (C-3, C-4, C-5, C-2′, C-3′, C-4′, C-5′,
C-3′′, C-4′′, C-5′′, C-2′′′, C-3′′′, C-4′′′, C-5′′′, C-3′′′′, C-4′′′′, C-5′′′′),
92.5 (Cq CCl3), 92.5 (Cq CCl3), 92.7 (Cq CCl3), 99.5, 99.7, 99.9,
99.9, 101.1 (C-1, C-1′, C-1′′, C-1′′′, C-1′′′′), 128.2-128.9 (CH
Arom, Cq Arom), 129.5-130.0 (CH Arom, Cq Arom), 132.9-133.5
(CH Arom), 161.4 (CdO TCA), 161.6 (CdO TCA), 161.8 (CdO
TCA), 165.1 (CdO Bz), 165.2 (CdO Bz), 165.6 (CdO Bz), 165.7
(CdO Bz), 169.7 (CdO COOMe), 170.7 (CdO COOMe), 172.1
(CdOCOOLev),205(CdOCOLev);HRMS:C98H127Cl9N6O34Si3
+ 2H+ requires 1166.2522, found 1166.2526.
22
foam (1.29 g, 72%). [R]D ) -26 (c ) 0.1, DCM); IR (neat):
1
827, 1070, 1521, 1716, 2098, 2341, 2359, 2860, 2933 cm-1; H
NMR (400 MHz, CDCl3): δ ) 0.84 (s, 9H, tBu), 0.88 (s, 9H, tBu),
0.90 (s, 9H, tBu), 1.03 (s, 9H, tBu), 1.79-1.82 (m, 2H, CH2
C3H6N3), 2.14 (s, 3H, CH3 Lev), 2.54-2.57 (m, 2H, CH2 Lev),
2.67-2.71 (m, 3H, H-6′′, CH2 Lev), 3.28 (dd, 1H, J ) 4.8 Hz, 9.6
Hz H-5′′), 3.34 (t, 2H, J ) 6.4 Hz, CH2 C3H6N3), 3.46 (dd, 1H, J
) 4.8 Hz, 9.6 Hz, H-5), 3.54-3.62 (m, 4H, H-2, H-4′′, H-6′′, CH2
C3H6N3), 3.84 (s, 3H, CH3 COOMe), 3.87-3.91 (m, 4H, H-4, H-6,
H-2′′, CH2 C3H6N3), 4.16-4.19 (m, 2H, H-6, H-5′), 4.70 (t, 1H, J
) 9.2 Hz, H-3), 4.33 (t, 1H, J ) 9.2 Hz, H-4′), 4.81 (d, 1H, J )
8.0 Hz, H-1), 4.97 (d, 1H, J ) 8.4 Hz, H-1′′), 5.02 (t, 1H, J ) 9.6
Hz, H-3′′), 5.21 (dd, 1H, J ) 4.4 Hz, 8.8 Hz, H-2′), 5.39 (d, 1H,
J ) 4.4 Hz, H-1′) 5.62 (t, 1H, J ) 9.2 Hz, H-3′), 6.91 (d, 1H, J )
8.8 Hz, NH), 7.01 (d, 1H, J ) 8.4 Hz, NH), 7.34-7.43 (m, 4H, H
Arom), 7.49-7.54 (m, 2H, H Arom), 7.91-7.93 (m, 4H, H
Arom);13C NMR (100 MHz): δ ) 19.6 (Cq tBu), 19.7 (Cq tBu),
22.3 (Cq tBu), 22.5 (Cq tBu), 26.6 (CH3 tBu), 26.6 (CH3 tBu), 27.1
(2 x CH3 tBu), 27.8 (CH2 Lev), 28.8 (CH2 C3H6N3), 29.6 (CH3
Lev), 37.9 (CH2 Lev), 47.7 (CH2 C3H6N3), 52.9 (CH3 COOMe),
55.5 (C-2′′), 57.3 (C-2), 64.9 (C-6′′), 65.9 (C-6), 66.4 (CH2 C3H6N3),
70.2 (C-5), 70.5 (C-5′′), 71.3 (C-3′), 74.0, 74.2, 74.3, 74.4 (C2′,
C-5′, C-3′′, C-4′′), 76.0 (C-4), 76.5 (C-4′), 77.8 (C-3), 92.3 (Cq
CCl3), 92.5 (Cq CCl3), 99.6 (C-1′), 99.9 (C-1), 100.9 (C-1′′),
128.2-128.7 (CH Arom, Cq Arom), 129.5-129.8 (CH Arom, Cq
Arom), 133.0-133.4 (CH Arom), 161.5 (CdO TCA), 161.7 (CdO
TCA), 165.0 (CdO Bz), 165.6 (CdO Bz), 170.2 (CdO COOMe),
172.0 (CdO COO Lev), 205.9 (CdO CO Lev); HRMS:
C61H83Cl6N5O21Si2 + H+ requires 1488.3323, found 1488.3330.
3-Azidopropyl (4,6-O-di-tert-butylsilylidene-2-deoxy-2-trichlo-
roacetamido-3-O-(methyl (2,3-di-O-benzoyl-4-O-(4,6-O-di-tert-bu-
tylsilylidene-2-deoxy-2-trichloroacetamido-ꢀ-D-glucopyranosyl)-ꢀ-
D-glucopyranosyl) uronate)-ꢀ-D-glucopyranoside (17). Trimer 16
(1.08 g, 0.72 mmol) was dissolved in a mixture of pyridine (6.4
mL) and AcOH (1.6 mL), after which hydrazine monohydrate (0.18
mL, 3.6 mmol) was added. The mixture was stirred for 15 min
and diluted with EtOAc (20 mL), 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
(PE, EtOAc) yielded 17 as a white foam (0.942 g, 94%). [R]D22
) -26 (c ) 0.1, DCM); IR (neat): 827, 1072, 1527, 1724, 2100,
2860, 2933 cm-1; 1H NMR (400 MHz, CDCl3): δ ) 0.85 (s, 9H,
tBu), 0.90 (s, 18H, tBu), 1.03 (s, 9H, tBu), 1.78-1.80 (m, 2H, CH2
C3H6N3), 2.65 (t, 1H, J ) 12.0 Hz, H-6′′), 2.93 (bs, 1H, OH),
3.22 (dd, 1H, J ) 4.8 Hz, 9.6 Hz H-5′′), 3.31-3.40 (m, 3H, H-3′′,
CH2 C3H6N3), 3.45-3.58 (m, 5H, H-2, H-5, H-2′′, H-6′′, CH2
C3H6N3), 3.74 (t, 1H, J ) 9.6 Hz, H-4′′), 3.84 (s, 3H, CH3
COOMe), 3.86-3.97 (m, 3H, H-4, H-6, CH2 C3H6N3), 4.11-4.20
(m, 2H, H-6, H-5′), 4.26-4.36 (m, 2H, H-3, H-4′), 4.82 (d, 1H, J
) 8.4 Hz, H-1), 4.98 (d, 1H, J ) 8.4 Hz, H-1′′), 5.24 (dd, 1H, J )
4.4 Hz, 8.8 Hz, H-2′), 5.36 (d, 1H, J ) 4.4 Hz, H-1′) 5.61 (t, 1H,
J ) 9.2 Hz, H-3′), 6.97 (d, 1H, J ) 8.0 Hz, NH), 7.04 (d, 1H, J )
7.6 Hz, NH), 7.34-7.42 (m, 4H, H Arom), 7.49-7.55 (m, 2H, H
Arom), 7.91-7.93 (m, 4H, H Arom);13C NMR (100 MHz): δ )
19.7 (Cq tBu), 19.8 (Cq tBu), 22.4 (Cq tBu), 22.7 (Cq tBu), 26.6
(CH3 tBu), 26.8 (CH3 tBu), 27.2 (CH3 tBu), 27.3 (CH3 tBu), 28.9
(CH2 C3H6N3), 47.8 (CH2 C3H6N3), 53.0 (CH3 COOMe), 57.6
Azidopropyl (4,6-O-di-tert-butylsilylidene-2-deoxy-2-trichloro-
acetamido-3-O-(methyl (2,3-di-O-benzoyl-4-O-(4,6-O-di-tert-
butylsilylidene-2-deoxy-2-trichloroacetamido-3-O-(methyl (2,3-di-
O-benzoyl-4-O-(4,6-O-di-tert-butylsilylidene-2-deoxy-2-trichloroac-
etamido-ꢀ-D-glucopyranosyl)-ꢀ-D-glucopyranosyl) uronate)-ꢀ-D-
glucopyranosyl)-ꢀ-D-glucopyranosyl) uronate)-ꢀ-D-glucopyranoside
(19). Pentamer 18 (1.635 g, 0.70 mmol) was dissolved in a mixture
of pyridine (8 mL) and AcOH (2 mL), after which hydrazine
monohydrate (35 µL, 3.58 mmol) was added. The mixture was
stirred for 30 min and diluted with EtOAc (20 mL), 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 (PE, EtOAc) yielded 19 as a white foam
4214 J. Org. Chem. Vol. 74, No. 11, 2009

Synthesis of Hyaluronic Acid Oligomers
(1.42 g, 91%). IR (neat): 828, 1072, 1527, 1719, 2098, 2829, 2933
cm-1; 1H NMR (400 MHz, CDCl3): δ ) 0.78 (s, 9H, tBu), 0.86 (s,
9H, tBu), 0.89 (m, 18H, tBu), 0.91 (s, 9H, tBu), 1.04 (s, 9H, tBu),
1.78-1.80 (m, 2H, CH2 C3H6N3), 2.65-2.67 (m, 2H, H-6′′′′, H-6′′),
2.93 (bs, OH), 3.20-3.25 (m, 2H), 3.33 (t, 2H, J ) 6.8 Hz, CH2
C3H6N3), 3.37-3.70 (m, 11H), 3.78-3.79 (m, 1H), 3.82 (s, 3H,
CH3 COOMe), 3.83 (s, 3H, CH3 COOMe), 3.86-3.97 (m, 5H),
4.06-4.19 (m, 5H), 4.26-4.41 (m, 4H), 4.82 (d, 1H, J ) 8.0 Hz),
4.96-5.04 (m, 3H), 5.09-5.12 (m, 1H), 5.22-5.25 (m, 1H),
5.33-5.41 (m, 2H), 5.53-5.60 (m, 2H), 6.88-6.93 (m, 2H, NH),
7.14 (d, 1H, J ) 7.6 Hz, NH), 7.33-7.42 (m, 8H, H Arom),
7.48-7.54 (m, 4H, H Arom), 7.88-7.92 (m, 8H, H Arom);13C
NMR (100 MHz): δ ) 19.5 (Cq tBu), 19.6 (Cq tBu), 22.4 (Cq tBu),
22.5 (Cq tBu), 26.6-26.8 (CH3 tBu), 27.0-27.3 (CH3 tBu), 28.9
(CH2 C3H6N3), 47.8 (CH2 C3H6N3), 52.7 (CH3 COOMe), 52.9 (CH3
COOMe), 57.1, 57.7, 57.7 (C-2, C-2′′, C-2′′′′), 65.1, 65.1, 66.0,
(C-6, C-6′′, C-6′′′′), 66.5 (CH2 C3H6N3), 69.8, 70.1, 70.3, 71.3, 71.5,
73.7, 74.3, 74.4, 74.7, 74.9, 75.8, 75.9, 76.3, 77.0, 77.8, 78.4, (C-
3, C-4, C-5, C-2′, C-3′, C-4′, C-5′, C-3′′, C-4′′, C-5′′, C-2′′′, C-3′′′,
C-4′′′, C-5′′′, C-3′′′′, C-4′′′′, C-5′′′′), 92.5 (Cq CCl3), 92.6 (Cq CCl3),
92.6 (Cq CCl3), 99.5, 99.6, 99.8, 99.8, 100.3 (C-1, C-1′, C-1′′, C-1′′′,
C-1′′′′), 128.2-128.9 (CH Arom, Cq Arom), 129.5-129.9 (CH
Arom, Cq Arom), 132.7-133.4 (CH Arom), 161.4 (CdO TCA),
161.7 (CdO TCA), 162.3 (CdO TCA), 165.0 (CdO Bz), 165.2
(CdO Bz), 165.6 (CdO Bz), 165.7 (CdO Bz), 169.7 (CdO
COOMe), 170.9 (CdO COOMe); HRMS: C93H121Cl9N6O32Si3 +
2K+ requires 1136.2117, found 1136.2108.
6, C-6′′, C-6′′′′, C-6′′′′′′), 66.6 (CH2 C3H6N3), 69.9, 70.4, 70.6, 71.4,
71.5, 71.6, 73.8, 74.4, 74.4, 74.4, 74.7, 74.8, 75.8, 75.9, 77.8, 78.2,
(C-3, C-4, C-5, C-2′, C-3′, C-4′, C-5′, C-3′′, C-4′′, C-5′′, C-2′′′,
C-3′′′, C-4′′′, C-5′′′, C-3′′′′, C-4′′′′, C-5′′′′, C-2′′′′′, C-3′′′′′, C-4′′′′′,
C-5′′′′′, C-3′′′′′′, C-4′′′′′′, C-5′′′′′′), 92.5 (Cq CCl3), 99.5, 99.6, 99.9,
100.0, 101.2 (C-1, C-1′, C-1′′, C-1′′′, C-1′′′′, C-1′′′′′, C-1′′′′′′),
128.3-128.9 (CH Arom, Cq Arom), 129.5-130.0 (CH Arom, Cq
Arom), 133.0-133.5 (CH Arom), 161.4 (CdO TCA), 161.4 (CdO
TCA), 161.6 (CdO TCA), 161.8 (CdO TCA), 165.1 (CdO Bz),
165.1 (CdO Bz), 165.2 (CdO Bz), 165.6 (CdO Bz), 165.7 (CdO
Bz), 165.7 (CdO Bz), 169.7 (CdO COOMe), 170.3 (CdO
COOMe), 170.8 (CdO COOMe), 172.1 (CdO COO Lev); HRMS:
C
135H171Cl12N7O47Si4 + 2H+ requires 1587.8349, found 1587.8345.
3-Azidopropyl (2-deoxy-2-amino-3-O-(4-O-(2-deoxy-2-amino-
ꢀ-D-glucopyranosyl)-ꢀ-D-glucopyranosyl) uronate)-ꢀ-D-glucopy-
ranoside (21). Fully protected trimer 16 (0.173 g, 0.116 mmol)
was dissolved in THF (2.3 mL) and Et3N/3HF (0.11 mL, 0.696
mmol) was added. After 2 h the mixture was diluted with EtOAc
and washed with NaHCO3(aq). The water layer was extracted twice
with EtOAc and the collected organic layers were dried over MgSO4
and concentrated in Vacuo. The resulting syrup was then dissolved
in THF (2 mL) and H2O (2 mL) and a 0.5 M solution of KOH in
H2O (1.62 mL, 0.812 mmol) was added stepwise (per 1 equiv.)
over a period of 10 h. The reaction mixture was stirred for 4 days
after which it was quenched with AcOH and concentrated in Vacuo.
The remaining solid was subsequently purified by gel filtration and
lyophilized 3 times yielding 21 as a white amorphous solid (28
1
mg, 48%). H NMR (600 MHz, CDCl3): δ ) 1.87-1.92 (m, 2H,
Azidopropyl (4,6-O-di-tert-butylsilylidene-2-deoxy-2-trichlo-
roacetamido-3-O-(methyl (2,3-di-O-benzoyl-4-O-(4,6-O-di-tert-
butylsilylidene-2-deoxy-2-trichloroacetamido-3-O-(methyl (2,3-
di-O-benzoyl-4-O-(4,6-O-di-tert-butylsilylidene-2-deoxy-2-
trichloroacetamido-3-O-(methyl (2,3-di-O-benzoyl-4-O-(4,6-O-
di-tert-butylsilylidene-2-deoxy-3-O-levulinoyl-2-trichloroacetamido-
ꢀ-D-glucopyranosyl)-ꢀ-D-glucopyranosyl) uronate)-ꢀ-D-glucopy-
ranosyl)-ꢀ-D-glucopyranosyl) uronate)-ꢀ-D-glucopyranosyl)-ꢀ-
D-glucopyranosyl) uronate)-ꢀ-D-glucopyranoside (20). Thio dimer
3 (0.542 g, 0.515 mmol) and pentamer acceptor 19 (0.922 g, 0.412
mmol) were coevaporated with toluene two times to remove traces of
water then dissolved in DCM (4.1 mL). NIS (0.116 g, 0.515 mmol)
was added and the mixture was stirred over activated 3 Å molsieves
for 30 min. The mixture was cooled to 0 °C before a catalytic amount
of TfOH (2 µL, 0.025 mmol) was added, then the mixture was allowed
to warm to RT. TLC analysis was complicated because of the viscosity
of the mixture as well as the similar polarities of donor and acceptor.
After allowing the reaction to run for approximately 4 h (this reaction
time was estimated based on the construction of the pentamer, which
took approximately 2 h), the reaction was quenched with Et3N. The
CH2 C3H6N3), 3.04 (dd, 1H, J ) 8.4, 10.2 Hz, H-2 or H-2′′), 3.08
(t, 1H, J ) 9 Hz, H-2 or H-2′′), 3.43 (t, 2H, J ) 6.6 Hz, CH2
C3H6N3), 3.45-3.50 (m, 4H), 3.61-3.67 (m, 4H), 3.69-3.76 (m,
4H), 3.79-3.82 (m, 1H, H-5′), 3.88-3.93 (m, 3H), 3.97-4.01 (m,
1H, CH2 C3H6N3), 4.63 (d, 1H, J ) 8.4 Hz, H-1 or H-1′ or H-1′′),
4.67 (d, 1H, J ) 8.4 Hz, H-1 or H-1′ or H-1′′), 4.74 (d, 1H, J )
8.4 Hz, H-1 or H-1′ or H-1′′);13C NMR (150 MHz): δ ) 28.2 (CH2
C3H6N3), 48.0 (CH2 C3H6N3), 55.1, 55.7 (C-2, C-2′′), 60.3, 60.4
(C-6, C-6′′), 67.6 (CH2 C3H6N3), 68.0, 69.4, 72.2, 72.7, 74.1, 74.6,
75.8, 76.3, 79.9 (C-3, C-4, C-5, C-2′, C-3′, C-4′, C-3′′, C-4′′, C-5′′),
82.8 (C-5′), 98.9, 99.8, 101.9 (C-1, C-1′, C-1′′), 174.9 (COOH);
HRMS: C21H37N5O15 + H+ requires 600.2359, found 600.2378.
3-Azidopropyl (2-deoxy-2-acetamido-3-O-(4-O-(2-deoxy-2-ac-
etamido-ꢀ-D-glucopyranosyl)-ꢀ-D-glucopyranosyl) uronate)-ꢀ-D-
glucopyranoside (22). Zwitterionic HA-trisaccharide 21 (17 mg,
0.028 mmol) was dissolved in MeOH (5 mL) and Ac2O (0.5 mL)
was added. After 4 h, this mixture was coevaporated three times
with toluene and concentrated in Vacuo. When NMR revealed an
additional methylester signal, the residue was dissolved in H2O and
LiOH (0.1 mL, 0.5 M) was added. The mixture was stirred for 2 h
and quenched with AcOH until neutral and concentrated in Vacuo.
The remaining solid was subsequently purified by gel filtration and
lyophilized 3 times yielding 22 as a white amorphous solid (19
reaction mixture was diluted with DCM and washed with NaHCO3 (aq)
.
The water layer was extracted twice with DCM and the collected
organic layers were dried over MgSO4 and concentrated in Vacuo.
Purification by size exclusion and column chromatography (PE,
EtOAc) yielded 20 as a white foam (0.76 g, 61%). IR (neat): 826,
1
mg, 99%). H NMR (400 MHz, CDCl3): δ ) 1.79-1.84 (m, 2H,
1028, 1068, 1707, 2098, 2860, 2934 cm-1; H NMR (400 MHz,
1
CH2 C3H6N3), 2.01 (s, 3H, CH3 Ac), 2.03 (s, 3H, CH3 Ac),
3.32-3.36 (m, 3H), 3.40-3.57 (m, 7H), 3.62-3.75 (m, 7H),
3.78-3.83 (m, 1H), 3.88-3.98 (m, 3H), 4.45 (d, 1H, J ) 6.7 Hz,
H-1 or H-1′ or H-1′′), 4.50 (d, 1H, J ) 8.4 Hz, H-1 or H-1′ or
H-1′′), 4.51 (d, 1H, J ) 8.4 Hz, H-1 or H-1′ or H-1′′);13C NMR
(100 MHz): δ ) 22.2 (CH3 Ac), 22.4 (CH3 Ac), 28.1 (CH2 C3H6N3),
47.7 (CH2 C3H6N3), 54.5, 55.3 (C-2, C-2′′), 60.5, 60.6 (C-6, C-6′′),
67.1 (CH2 C3H6N3), 68.5, 69.6, 72.4, 73.5, 73.8, 75.3, 75.8, 76.3,
79.8, 82.2 (C-3, C-4, C-5, C-2′, C-3′, C-4′, C-3′′, C-4′′, C-5′, C-5′′),
100.7, 100.9, 103.1 (C-1, C-1′, C-1′′), 174.2, 174.6, 174.8 (CdO
Ac, COOH); HRMS: C25H41N5O17 + H+ requires 684.2570, found
684.2573.
CDCl3): δ ) 0.73 (s, 9H, tBu), 0.74 (s, 9H, tBu), 0.85 (s, 9H, tBu),
0.89 (s, 36H, tBu), 1.03 (s, 9H, tBu), 1.78-1.81 (m, 2H, CH2
C3H6N3), 2.13 (s, 3H, CH3 Lev), 2.54-2.57 (m, 2H, CH2 Lev),
2.64-2.71 (m, 5H, H-6′′, H-6′′′′, H-6′′′′′′), 3.19-3.22 (m, 2H),
3.23-3.35 (m, 3H), 3.38-3.63 (m, 12H), 3.81 (s, 6H, CH3
COOMe), 3.83 (s, 3H, CH3 COOMe), 3.85-3.96 (m, 5H),
4.07-4.18 (m, 7H), 4.28-4.36 (m, 4H), 4.82 (d, 1H, J ) 8.0 Hz),
4.96-5.04 (m, 4H), 5.09-5.13 (m, 2H), 5.22-5.25 (m, 1H),
5.31-5.39 (m, 3H), 5.51-5.59 (m, 3H), 6.88-6.98 (m, 4H, NH),
7.33-7.42 (m, 12H, H Arom), 7.48-7.55 (m, 6H, H Arom),
7.87-7.93 (m, 12H, H Arom);13C NMR (100 MHz): δ ) 19.6 (Cq
tBu), 19.7 (Cq tBu), 22.4 (Cq tBu), 22.6 (Cq tBu), 26.6-26.7 (CH3
tBu), 27.1-27.2 (CH3 tBu), 28.0 (CH2 Lev), 28.9 (CH2 C3H6N3),
29.6 (CH3 Lev), 38.1 (CH2 Lev), 47.9 (CH2 C3H6N3), 52.8 (CH3
COOMe), 52.8 (CH3 COOMe), 53.0 (CH3 COOMe), 55.6, 57.0,
57.3, 57.8 (C-2, C-2′′, C-2′′′′, C-2′′′′′′), 65.1, 65.1, 65.2, 66.1, (C-
Azidopropyl (2-deoxy-2-amino-3-O-(4-O-(2-deoxy-2-amino-
3-O-(4-O-(2-deoxy-2-amido-ꢀ-D-glucopyranosyl)-ꢀ-D-glucopyra-
nosyl) uronate)-ꢀ-D-glucopyranosyl)-ꢀ-D-glucopyranosyl) uronate)-
ꢀ-D-glucopyranoside (23). Fully protected HA pentamer 18 (0.289
g, 0.124 mmol) was dissolved in THF (3 mL) and Et3N/3HF (0.121
J. Org. Chem. Vol. 74, No. 11, 2009 4215

Dinkelaar et al.
mL, 0.741 mmol) was added. After 6 h, the mixture was diluted
with EtOAc and was washed with NaHCO3(aq). The water layer
was extracted twice with EtOAc, the collected organic layers were
dried over MgSO4 and concentrated in Vacuo. The resulting syrup
was then dissolved in THF (3 mL) and H2O (3 mL) and a 0.5 M
solution of KOH in H2O (2.73 mL, 1.36 mmol) was added stepwise
(per 1 equiv) over a period of 72 h. The reaction mixture was stirred
for 4 days after which it was quenched with AcOH and concentrated
in Vacuo. The remaining solid was subsequently purified by gel
filtration and lyophilized 3 times yielding 23 as a white amorphous
water layer was extracted twice with EtOAc the collected organic
layers were dried over MgSO4 and concentrated in Vacuo. The
resulting syrup was then dissolved in THF (1.6 mL) and H2O (1.6
mL) and a 0.5 M solution of KOH in H2O (0.164 mL, 1.23 mmol)
was added stepwise (per 1 equiv) over a period of 10 h. The reaction
mixture was stirred for 4 days, after which it was quenched with
AcOH and concentrated in Vacuo. The remaining solid was
subsequently purified by gel filtration and lyophilized 3 times
1
yielding 25 as a white amorphous solid (64 mg, 59%). H NMR
(600 MHz, CDCl3): δ ) 1.86-1.90 (m, 2H, CH2 C3H6N3), 2.93 (t,
1H, J ) 9.6 Hz, H-2 or H-2′′ or H-2′′′′ or H-2′′′′′′), 2.98 (t, 1H, J
) 10.2 Hz, H-2 or H-2′′ or H-2′′′′ or H-2′′′′′′), 3.08 (t, 2H, J ) 9.6
Hz, H-2 or H-2′′ or H-2′′′′ or H-2′′′′′′), 3.41-3.49 (m, 11H), 3.55
(t, 1H, J ) 9.6 Hz), 3.59-3.80 (m, 18H), 3.87-3.91 (m, 7H),
3.96-3.99 (m, 1H, CH2 C3H6N3), 4.50 (d, 1H, J ) 8.4 Hz, H-1 or
H-1′ or H-1′′ or H-1′′′ or H-1′′′′ or H-1′′′′′, or h-1′′′′′′), 4.62-4.63
(M, 4H, H-1 or H-1′ or H-1′′ or H-1′′′ or H-1′′′′ or H-1′′′′′, or
h-1′′′′′′), 4.64 (d, 1H, J ) 8.4 Hz, H-1 or H-1′ or H-1′′ or H-1′′′ or
H-1′′′′ or H-1′′′′′, or h-1′′′′′′), 4.68 (d, 1H, J ) 8.4 Hz, H-1 or H-1′
or H-1′′ or H-1′′′ or H-1′′′′ or H-1′′′′′, or h-1′′′′′′); 13C NMR (150
MHz): δ ) 28.2 (CH2 C3H6N3), 48.0 (CH2 C3H6N3), 55.3, 55.3,
55.5, 55.8 (C-2, C-2′′, C-2′′′′, C-2′′′′′′), 60.3, 60.3, 60.4 (C-6, C-6′′,
C-6′′′′, C-6′′′′′′), 67.5 (CH2 C3H6N3), 67.9, 67.9, 68.1, 69.5, 72.6,
72.8, 74.2, 74.2, 74.7, 74.9, 75.0, 75.8, 75.8, 76.2, 80.1, 80.2 (C-3,
C-4, C-5, C-2′, C-3′, C-4′, C-3′′, C-4′′, C-5′′, C-2′′′, C-3′′′, C-4′′′,
C-3′′′′, C-4′′′′, C-5′′′′, C-2′′′′′, C-3′′′′′, C-4′′′′′, C-3′′′′′′, C-4′′′′′′,
C-5′′′′′′), 83.4, 83.5, 84.7 (C-5′, C-5′′′, C-5′′′′′), 99.6, 99.6, 100.3,
101.2, 102.2, 102.2, 102.5 (C-1, C-1′, C-1′′, C-1′′′, C-1′′′′, C-1′′′′′,
C-1′′′′′′), 174.7, 174.7, 174.8 (COOH); HRMS: C45H75N7O35 + H+
requires 1274.4377, found 1274.4395.
Azidopropyl (2-deoxy-2-acetamido-3-O-(4-O-(2-deoxy-2-aceta-
mido-3-O-(4-O-(2-deoxy-2-acetamido-3-O-(4-O-(2-deoxy-2-acetamido-
ꢀ-D-glucopyranosyl)-ꢀ-D-glucopyranosyl) uronate)-ꢀ-D-glucopyr-
anosyl)-ꢀ-D-glucopyranosyl) uronate)-ꢀ-D-glucopyranosyl)-ꢀ-D-
glucopyranosyl) uronate)-ꢀ-D-glucopyranoside (26). Zwitterionic
HA-heptasaccharide 25 (11 mg, 0.0089 mmol) was dissolved in
MeOH (5 mL) and Ac2O (0.5 mL) was added. After 4 h, this
mixture was coevaporated three times with toluene and concentrated
in Vacuo. When NMR revealed additional methyl ester signals, the
residue was dissolved in H2O and LiOH (0.1 mL, 0.5 M) was added.
The mixture was stirred for 2 h and quenched with AcOH until
neutral and concentrated in Vacuo. The remaining solid was
subsequently purified by gel filtration and lyophilized 3 times
yielding 26 as a white amorphous solid (10 mg, 78%).1H NMR
(400 MHz, CDCl3): δ ) 1.80-1.84 (m, 2H, CH2 C3H6N3), 2.00
(s, 6H, CH3 Ac), 2.01 (s, 6H, CH3 Ac), 2.03 (s, 3H, CH3 Ac),
3.30-3.37 (m, 5H), 3.43-3.58 (m, 13H), 3.60-3.72 (m, 17H),
3.74-3.84 (m, 3H), 3.88-3.95 (m, 4H), 3.96-3.98 (m, 1H),
4.43-4.54 (m, 7H, H-1, H-1′, H-1′′, H-1′′′, H-1′′′′, H-1′′′′′, H-1′′′′′′);
13C NMR (100 MHz): δ ) 22.2 (CH3 Ac), 22.4 (CH3 Ac), 22.4
(CH3 Ac), 28.0 (CH2 C3H6N3), 47.7 (CH2 C3H6N3), 54.3, 54.3, 54.5,
55.3 (C-2, C-2′′, C-2′′′′, C-2′′′′′′), 60.5, 60.6 (C-6, C-6′′, C-6′′′′,
C-6′′′′′′), 67.1 (CH2 C3H6N3), 68.4, 68.5, 69.6, 72.4, 73.5, 73.8,
75.3, 75.8, 76.2, 76.4, 79.7, 79.9 (C-3, C-4, C-5, C-2′, C-3′, C-4′,
C-3′′, C-4′′, C-5′′, C-2′′′, C-3′′′, C-4′′′, C-3′′′′, C-4′′′′, C-5′′′′, C-2′′′′′,
C-3′′′′′, C-4′′′′′, C-3′′′′′′, C-4′′′′′′, C-5′′′′′′), 82.1, 82.5 (C-5′, C-5′′′,
C-5′′′′′), 100.6, 100.9, 103.1 (C-1, C-1′, C-1′′, C-1′′′, C-1′′′′, C-1′′′′′,
C-1′′′′′′), 174.1, 174.2, 174.6, 174.8, 174.9 (CdO Ac, COOH);
HRMS: C53H83N7O39 + H+ requires 1442.4799, found 1442.4819.
1
solid (51 mg, 42%). H NMR (600 MHz, CDCl3): δ ) 1.86-1.91
(m, 2H, CH2 C3H6N3), 3.01-3.06 (m, 2H, H-2 or H-2′′ or H-2′′′′),
3.14-3.18 (m, 1H, H-2 or H-2′′ or H-2′′′′), 3.41-3.50 (m, 8H), 3.60
(t, 1H, J ) 9 Hz), 3.63-3.67 (m, 4H), 3.69-3.78 (m, 7H), 3.86-3.93
(m, 6H), 3.96-4.00 (m, 1H, CH2 C3H6N3), 4.59 (d, 1H, J ) 8.4 Hz,
H-1 or H-1′ or H-1′′ or H-1′′′ or H-1′′′′), 4.65 (d, 1H, J ) 8.4 Hz, H-1
or H-1′ or H-1′′ or H-1′′′ or H-1′′′′), 4.66 (d, 1H, J ) 8.4 Hz, H-1 or
H-1′ or H-1′′ or H-1′′′ or H-1′′′′), 4.70 (d, 1H, J ) 9.0 Hz, H-1 or
H-1′ or H-1′′ or H-1′′′ or H-1′′′′), 4.73 (d, 1H, J ) 8.4 Hz, H-1 or
H-1′ or H-1′′ or H-1′′′ or H-1′′′′);13C NMR (150 MHz): δ ) 28.2
(CH2 C3H6N3), 48.0 (CH2 C3H6N3), 55.0, 55.2, 55.7 (C-2, C-2′′, C-2′′′′),
60.3, 60.3, 60.4 (C-6, C-6′′, C-6′′′′), 67.6 (CH2 C3H6N3), 67.8, 68.0,
69.5, 72.2, 72.6, 72.7, 74.1, 74.5, 74.7, 75.8, 75.9, 76.2, 80.0, 80.1
(C-3, C-4, C-5, C-2′, C-3′, C-4′, C-3′′, C-4′′, C-5′′, C-2′′′, C-3′′′, C-4′′′,
C-3′′′′, C-4′′′′, C-5′′′′), 82.4, 83.3 (C-5′, C-5′′′), 99.1, 99.5, 100.2, 101.8,
102.1 (C-1, C-1′, C-1′′, C-1′′′, C-1′′′′), 174.8, 174.8 (COOH); HRMS:
C33H56N6O25 + H+ requires 937.3368, found 937.3376.
Azidopropyl (2-deoxy-2-acetamido-3-O-(4-O-(2-deoxy-2-ac-
etamido-3-O-(4-O-(2-deoxy-2-acetamido-ꢀ-D-glucopyranosyl)-ꢀ-
D-glucopyranosyl) uronate)-ꢀ-D-glucopyranosyl)-ꢀ-D-glucopyr-
anosyl) uronate)-ꢀ-D-glucopyranoside (24). Zwitterionic HA-
pentasaccharide 23 (12 mg, 0.013 mmol) was dissolved in MeOH
(5 mL) and Ac2O (0.5 mL) was added. After 4 h, this mixture was
coevaporated three times with toluene and concentrated in Vacuo.
When NMR revealed additional methyl ester signals, the residue
was dissolved in H2O and LiOH (0.1 mL, 0.5 M) was added. The
mixture was stirred for 2 h and quenched with AcOH until neutral
and concentrated in Vacuo. The remaining solid was subsequently
purified by gel filtration and lyophilized 3 times yielding 24 as a
white amorphous solid (10 mg, 74%).1H NMR (400 MHz, CDCl3):
δ ) 1.84-1.88 (m, 2H, CH2 C3H6N3), 2.05 (s, 6H, CH3 Ac), 2.07
(s, 3H, CH3 Ac), 3.37-3.40 (m, 4H), 3.47-3.62 (m, 10H),
3.69-3.86 (m, 14H), 3.92-4.12 (m, 4H), 4.48 (d, 1H, J ) 7.6 Hz,
H-1 or H-1′ or H-1′′ or H-1′′′ or H-1′′′′), 4.49 (d, 1H, J ) 8.0 Hz,
H-1 or H-1′ or H-1′′ or H-1′′′ or H-1′′′′), 4.54 (d, 1H, J ) 8.4 Hz,
H-1 or H-1′ or H-1′′ or H-1′′′ or H-1′′′′), 4.55 (d, 1H, J ) 9.6 Hz,
H-1 or H-1′ or H-1′′ or H-1′′′ or H-1′′′′), 4.57 (d, 1H, J ) 8.8 Hz,
H-1 or H-1′ or H-1′′ or H-1′′′ or H-1′′′′);13C NMR (100 MHz): δ
) 22.2 (CH3 Ac), 22.4 (CH3 Ac), 22.5 (CH3 Ac), 28.1 (CH2
C3H6N3), 47.8 (CH2 C3H6N3), 54.3, 54.6, 55.4 (C-2, C-2′′, C-2′′′′),
60.5, 60.5, 60.7 (C-6, C-6′′, C-6′′′′), 67.2 (CH2 C3H6N3), 68.4, 68.6,
69.7, 72.5, 72.5, 73.6, 73.9, 75.4, 75.9, 76.3, 76.4, 79.8, 80.0, 82.2,
82.6 (C-3, C-4, C-5, C-2′, C-3′, C-4′, C-3′′, C-4′′, C-5′′, C-2′′′,
C-3′′′, C-4′′′, C-3′′′′, C-4′′′′, C-5′′′′), 100.6, 100.7, 101.0, 103.1,
103.1 (C-1, C-1′, C-1′′, C-1′′′, C-1′′′′), 174.1, 174.2, 174.6, 174.9,
174.9 (CdO Ac, COOH); HRMS: C39H62N6O28 + H+ requires
1063.3685, found 1063.3694.
Azidopropyl (2-deoxy-2-amino-3-O-(4-O-(2-deoxy-2-amino-3-
O-(4-O-(2-deoxy-2-amino-3-O-(4-O-(2-deoxy-2-amino-ꢀ-D-glucopy-
ranosyl)-ꢀ-D-glucopyranosyl) uronate)-ꢀ-D-glucopyranosyl)-ꢀ-D-
glucopyranosyl) uronate)-ꢀ-D-glucopyranosyl)-ꢀ-D-glucopyranosyl)
uronate)-ꢀ-D-glucopyranoside (25). Fully protected HA heptamer
20 (0.261 g, 0.082 mmol) was dissolved in THF (1.6 mL) and Et3N/
3HF (0.107 mL, 0.656 mmol) was added. After 2 h the mixture
was diluted with EtOAc and was washed with NaHCO3(aq). The
Supporting Information Available: General experimental
and NMR spectra of all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO9003713
4216 J. Org. Chem. Vol. 74, No. 11, 2009