Chemical synthesis of a hyaluronic acid decasaccharide

Article abstract of DOI:10.1021/jo9016925

(Chemical Equation Presented) The chemical synthesis of a hyaluronic acid decasaccharide using the preactivation-based chemoselective glycosylation strategy is described. Assembly of large oligosaccharides is generally challenging due to the increased difficulties in both glycosylation and deprotection. Indeed, the same building blocks previously employed for hyaluronic acid hexasaccharide syntheses failed to yield the desired decasaccharide. After extensive experimentation, the decasaccharide backbone was successfully constructed with an overall yield of 37% from disaccharide building blocks. The trichloroacetyl group was used as the nitrogen protective group for the glucosamine units, and the addition of TMSOTf was found to be crucial to suppress the formation of trichloromethyl oxazoline side product and enable high glycosylation yield. For deprotections, the combination of a mild basic condition and the monitoring methodology using ¹H NMR allowed the removal of all base-labile protective groups, which facilitated the generation of the fully deprotected HA decasaccharide.

Full text of DOI:10.1021/jo9016925

pubs.acs.org/joc
Chemical Synthesis of a Hyaluronic Acid Decasaccharide
Xiaowei Lu, Medha N. Kamat, Lijun Huang,† and Xuefei Huang*
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824. †Current address:
Anatrace, Inc., 434 W. Dussel Dr., Maumee, OH 43537.
xuefei@chemistry.msu.edu
Received August 3, 2009
The chemical synthesis of a hyaluronic acid decasaccharide using the preactivation-based chemose-
lective glycosylation strategy is described. Assembly of large oligosaccharides is generally challenging
due to the increased difficulties in both glycosylation and deprotection. Indeed, the same building
blocks previously employed for hyaluronic acid hexasaccharide syntheses failed to yield the desired
decasaccharide. After extensive experimentation, the decasaccharide backbone was successfully
constructed with an overall yield of 37% from disaccharide building blocks. The trichloroacetyl
group was used as the nitrogen protective group for the glucosamine units, and the addition of
TMSOTf was found to be crucial to suppress the formation of trichloromethyl oxazoline side product
and enable high glycosylation yield. For deprotections, the combination of a mild basic condition and
the monitoring methodology using 1H NMR allowed the removal of all base-labile protective groups,
which facilitated the generation of the fully deprotected HA decasaccharide.
Introduction
promote angiogenesis.6,7 HA is the major ligand of CD44, a
transmembrane protein involved in cell-cell, cell-matrix
interactions and signal transduction.3 Evidence is accumu-
lating suggesting that the size of HA fragments recognized by
CD44 provides a physiologically important switch between
its adhesion and signaling functions.8 Binding of HA poly-
mers to CD44 usually leads to cell adhesion9,10 rather than
activation. In contrast, recognition of HA fragments instead
of the polymer leads to CD44 signaling, which is involved in
diseases such as cancer and inflammation.11-14 CD44 re-
quires at least an HA hexasaccharide sequence for binding,
Hyaluronic acid (HA), a component of the extracellular
matrix and a member of the glycosaminoglycan family,
contains linear tandem disaccharide repeats of D-glucuronic
acid and 2-deoxy-2-N-acetyl-D-glucose [β-D-GlcpA-(1f3)-
β-D-GlcpNAc-(1f4)-].1 HA can interact with a wide variety
of cell surface receptors, including the lymphatic vessel
endothelial receptor, CD44, TNF-simulated gene-6, and
HA receptor for endocytosis.2-4 The biological activities of
HA can be length dependent.5 For example, HA polymers
have been found to be antiangiogenic, while its oligomers
(8) Aruffo, A. J. Clin. Invest. 1996, 98, 2191–2192.
(9) DeGrendele, H. C.; Estess, P.; Picker, L. J.; Siegelman, M. H. J. Exp.
Med. 1996, 183, 1119–1130.
(1) Lapcik, L. Jr.; Lapcik, L.; De Smedt, S.; Demeester, J.; Chabrecek, P.
Chem. Rev. 1998, 98, 2663–2684.
(2) Harris, E. N.; Weigel, P. H. Glycobiology 2008, 18, 638–648 and
references cited therein.
(10) Jalkanen, S.; Bargatze, R. F.; de los Toyos, J.; Butcher, E. C. J. Cell
Biol. 1987, 105, 983–990.
(3) Ponta, H.; Sherman, L.; Herrlich, P. A. Nat. Rev. Cell Biol. 2003, 4,
33–45.
(4) Prevo, R.; Banerji, S.; Ferguson, D. J. P.; Clasper, S.; Jackson, D. G.
J. Biol. Chem. 2001, 276, 19420–19430.
(11) Jiang, D.; Liang, J.; Fan, J.; Yu, S.; Chen, S.; Luo, Y.; Prestwich,
G. D.; Mascarenhas, M. M.; Garg, H. G.; Quinn, D. A.; Homer, R. J.;
Goldstein, D. R.; Bucala, R.; Lee, P. J.; Medzhitov, R.; Noble, P. W. Nat.
Med. 2005, 11, 1173–1179.
(5) Asari, A. GlycoforumHyaluronan Today 2005, http://www.glycoforum.
gr.jp/science/hyaluronan/hyaluronanE.html.
(12) Misra, S.; Ghatak, S.; Zoltan-Jones, A.; Toole, B. P. J. Biol. Chem.
2003, 278, 25285–25288.
(6) West, D. C.; Fan, T.-P. D. In New Angiotherapy; Fan, T.-P. D., Kohn,
E. C., Eds.; Humana Press: Totowa, NJ, 2002; pp 177-188.
(7) West, D. C.; Hampson, I. N.; Arnold, F.; Kumar, S. Science 1985, 228,
1324–1326.
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38020.
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J. Clin. Invest. 1996, 98, 2403–2413.
7608 J. Org. Chem. 2009, 74, 7608–7617
Published on Web 09/21/2009
DOI: 10.1021/jo9016925
r
2009 American Chemical Society

Lu et al.
JOCFeatured Article
SCHEME 1. Previous Synthesis of HA Hexasaccharide 3
SCHEME 2
and only a decasaccharide or higher can effectively compete
with polymeric HA.15 Therefore, access to synthetic HA with
variable length and sequence can greatly facilitate the estab-
lishment of its structure-activity relationship.5
HA can be synthesized via either enzymatic or chemical
methods.16 Based on the understanding of HA biosynthetic
and catabolism pathways, innovative studies have been
carried out utilizing HA synthases or hyaluronidases to
acquire synthetic HA.17-19 While impressive synthetic effi-
ciency has been achieved, the inherent substrate specificities
of enzymes limit the structural diversity of HA analogues
that can be generated. Chemical synthesis can thus comple-
ment the enzymatic approaches to create greater varieties of
HA structures.
and heptasaccharide.27 Previously, our group has developed
a synthetic strategy to acquire HA oligosaccharides ranging
from di- to hexasaccharides.21 With the fascinating biologi-
cal activities of longer HA oligosaccharides, we became
interested in pursuing their chemical synthesis, which is not
a trivial extension of shorter oligomers because elongation of
the sugar sequences can create significant new challenges in
both glycosylation and deprotection. Herein, we report our
results on overcoming the obstacles for chemical synthesis of
an HA decasaccharide.
Results and Discussion
The design of a successful route to HA oligosaccharides
must take into account three factors: (1) stereochemical
control in glycosylation; (2) introduction of the glucuronic
acid; and (3) protective groups for the glucosamine nitrogen
moieties. In our previous hexasaccharide synthesis, we used
two key building blocks 1 and 2, which led to high yield of
hexasaccharide 3 in one pot (Scheme 1).21 The benzoyl (Bz)
and phthalimido (Phth) groups were crucial to facilitate the
formation of 1,2-trans glycosidic linkages. Due to its high
inherent reactivities,28 glucoside building block 1 was uti-
lized as a glucuronic acid surrogate.21 The p-methoxybenzyl
(PMB) groups in 3 masking the glucoside 6-O-position were
selectively removed by 2,3-dichloro-5,6-dicyano-1,4-benzo-
quinone (DDQ) oxidation, and the resulting triol was suc-
cessfully oxidized and deprotected producing HA
hexasaccharide. Following the success of hexasaccharide
synthesis, our initial attempt toward HA decasaccharide
hinged upon the usage of monosaccharides 1 and 2, from
which decasaccharide 4 was assembled. However, removal of
the five PMB groups in 4 turned out to be very problematic
Chemical syntheses of HA oligomers have been performed
with the majority of studies focusing on di- and
hexasaccharides.16,20-25 The longest HA oligosaccharides
chemically assembled to date are an HA octasaccharide26
€
(15) Tammi, R.; MacCallum, D.; Hascall, V. C.; Pienimaki, J. P.;
Hyttinen, M.; Tammi, M. J. Biol. Chem. 1998, 273, 28878–28888.
(16) Huang, L.; Lu, X.; Huang, X. In Chemical Glycobiology; Chen, X.,
Halcomb, R. L., Wang, P. G., Eds.; ACS Symp. Ser. 990; American Chemical
Society: Washington, DC, 2008; pp 29-53 and references cited therein.
(17) DeAngelis, P. L.; Oatman, L. C.; Gay, D. F. J. Biol. Chem. 2003, 278,
35199–35203 and references cited therein.
(18) Kobayashi, S.; Morii, H.; Itoh, R.; Kimura, S.; Ohmae, M. J. Am.
Chem. Soc. 2001, 123, 11825–11826.
(19) Luca, C. D.; Lansing, M.; Martini, I.; Crescenzi, F.; Shen, G.-J.;
O’Regan, M.; Wong, C.-H. J. Am. Chem. Soc. 1995, 117, 5869–5870.
(20) Dinkelaar, J.; Codee, J. D. C.; van den Bos, L. J.; Overkleeft, H. S.;
van Boom, J. H.; van der Marel, G. A. J. Org. Chem. 2007, 72, 5737–5742.
(21) Huang, L.; Huang, X. Chem.;Eur. J. 2007, 13, 529–540.
(22) Palmacci, E. R.; Seeberger, P. H. Tetrahedron 2004, 60, 7755–7766.
(23) Iyer, S. S.; Rele, S. M.; Baskarana, S.; Chaikof, E. L. Tetrahedron
2003, 59, 631–638.
(24) Yeung, B. K. S.; Chong, P. Y. C.; Petillo, P. A. In Glycochemistry.
Principles, Synthesis, and Applications; Wang, P. G., Bertozzi, C. R., Eds.;
Marcel Dekker, Inc.: New York, 2001; pp 425-492.
(25) Halkes, K. M.; Slaghek, T.; Hypponen, T. K.; Kruiskamp, P. H.;
Ogawa, T.; Kamerling, J. P.; Vliegenthart, J. F. G. Carbohydr. Res. 1998,
309, 161–174.
(27) Dinkelaar, J.; Gold, H.; Overkleeft, H. S.; Codee, J. D. C.; van der
Marel, G. A. J. Org. Chem. 2009, 74, 4208–4216.
(28) Zeng, Y.; Wang, Z.; Whitfield, D.; Huang, X. J. Org. Chem. 2008, 73,
7952–7962.
(26) Blatter, G.; Jacquinet, J.-C. Carbohydr. Res. 1996, 288, 109–125.
J. Org. Chem. Vol. 74, No. 20, 2009 7609

Lu et al.
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SCHEME 3. Retrosynthetic Scheme toward HA Decasaccharide 9
SCHEME 4. Syntheses of Monosaccharide Building Blocks 14
This prompted us to abandon the Phth group and utilize the
trichloroacetyl (TCA) group26,27 as an alternative protec-
tive group for nitrogen.
and 15a
Based on these considerations, a new retrosynthetic route
toward HA decasaccharide 9 is designed (Scheme 3). The
fully protected decasaccharide 10 will be assembled from
disaccharides 11, 12, and 13, which in turn will be derived
from monosaccharide building blocks 1, 14, and 15.
Glucoside donor 1 was prepared according to a literature
procedure,21 and the syntheses of glucosamine building
blocks 14 and 15 are outlined in Scheme 4. Peracetylated
N-trichloroacetylglucosamine 1626 was transformed into
the corresponding 1-β-p-tolyl thioglycoside 17 and the 1-β-
methoxy derivative 18. Compound 17 was then deacetylated
and protected with a benzylidene group to afford 14, whereas
compound 18 was protected by benzylidene to give 15.
With the monosaccharides in hand, disaccharide synthesis
was performed. Preactivation of glucoside donor 1 by the
reagent combination of silver triflate (AgOTf) and p-toluene-
sulfenyl chloride (p-TolSCl)21,32 at -78 °C was performed
followed bythe addition of acceptor 14and a bulky base 2,4,6-
tri-tert-butylpyrimidine (TTBP)33 leading to disaccharide 19
in 80% yield. The primary hydroxyl group in 19 was liberated
by treatment with DDQ and oxidized by pyridinium dichro-
mate (PDC) in dimethylformamide (DMF) to afford its
carboxylic acid derivative, which was protected as a benzyl
ester (disaccharide 11) in 65% yield for the three steps.
Subsequent removal of the TBS group led to the disaccharide
acceptor 12 bearing a free secondary hydroxyl group
(Scheme 5a). Disaccharide 13 was constructed through a
similar sequence from 1 and 15 in an overall yield of 48%
for the five steps (Scheme 5b).
aKey: (a) p-TolSH, BF3 OEt2, DCM; (b) (i) HBr/AcOH, (ii) Ag2CO3,
CaSO4, MeOH; (c) NaOMe/MeOH; (d) Ph(OMe)2, camphorsulfonic
acid.
3
(Scheme 2a). The previously employed DDQ oxidation21
decomposed the oligosaccharide. Cerium ammonium nitrate
(CAN) oxidation29 or the combination of p-tolyl sulfonam-
ide and triflic acid30 also failed to lead to the desired pentaol.
To circumvent this problem, the 6-hydroxyl group liberation
and oxidation state adjustment were performed on disac-
charide 6 (Scheme 2b). DDQ-mediated PMB removal fol-
lowed by primary hydroxyl oxidation and benzyl ester
formation produced disaccharide 7 containing the glucuro-
nic acid moiety. Condensations of the resulting disaccharides
afforded decasaccharide 8 with the glucuronic acids already
installed, which set the stage for deprotection. Although it
has been shown previously that Phth groups could be depro-
tected in molecules containing carboxylic esters,21,25,31 despite
repeated trials and screening of a variety of reagents and
reaction conditions, the conditions required to remove all
five Phth groups in 7 turned out to be incompatible with
the five carboxylic ester moieties present and vice versa.
Following the same glycosylation protocol as used in the
preparation of disaccharide 19, coupling of disaccharide
(29) Wuts, P. G. M.; Greene, T. W. Protective Groups in Organic Synth-
esis, 4th ed.; Wiley-Interscience: New York, 2006.
(30) Hinklin, R. J.; Kiessling, L. L. Org. Lett. 2002, 4, 1131–1133.
(31) Sun, B.; Srinivasan, B.; Huang, X. Chem.;Eur. J. 2008, 14, 7072–
7081.
(32) Huang, X.; Huang, L.; Wang, H.; Ye, X.-S. Angew. Chem., Int. Ed.
2004, 43, 5221–5224.
(33) Crich, D.; Smith, M.; Yao, Q.; Picione, J. Synthesis 2001, 323–326.
7610 J. Org. Chem. Vol. 74, No. 20, 2009

Lu et al.
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SCHEME 5. Assembly of Disaccharide Building Blocks
the final coupling of 25 (1.25 equiv) and 28 (1 equiv) only gave
decasaccharide 10 in 10% yield, with the majority of 25
converted to oxazoline 27 (85% yield, Table 1, entry 2). The
substantial increase of the amounts of oxazoline side products
is presumably because with the increasing sizes of the glycosyl
donor and acceptor, the compounds become less reactive
toward glycosylation (Scheme 6b, pathway a), which in turn
favors the competing reaction of oxazoline formation even in
the absence of base (Scheme 6b, pathway b).
SCHEME 6
donor 11 with acceptor 12 was attempted (Scheme 6a).
However, no desired tetrasaccharide was obtained with full
recovery of acceptor 12. Oxazoline derivative 20 (3JH1-H2
=
8.0 Hz34,35) was isolated from the reaction in quantitative
yield with the possible mechanism for its formation depicted
in Scheme 6b. Neighboring group participation by the TCA
group through attachment of its carbonyl oxygen to the
oxacarbenium ion forms the oxazolinium ion intermediate
22, which can react with an acceptor to afford the expected
1,2-trans-glycoside 23 (pathway a, Scheme 6b). Alterna-
tively, deprotonation of the oxazolinium ion produces the
stable trichlorooxazoline 24 (pathway b). Following this
mechanism, the failure of glycosylation could be attributed
to the addition of TTBP,27 which is used to scavenge the
triflic acid accumulated upon productive glycosylation.
Therefore, a second-generation glycosylation protocol was
adopted by omitting TTBP from the reaction. Following this
protocol, 11 (1 equiv) was condensed with 12 (1 equiv) to
produce tetrasaccharide 25 in 55% yield with 40% oxazoline
byproduct 20 (Table 1, entry 2). The next condensation of
tetrasaccharide donor 25 (1 equiv) with disaccharide acceptor
13 gave hexasaccharide 26 in 40% yield and oxazoline 27
(50% yield). Subsequent cleavage of the TBS group in 26 gave
the hexasaccharide acceptor28in90% yield. Disappointingly,
In order to suppress the amount of oxazoline, exogenous
triflic acid (∼0.4 equiv) was introduced to the reaction
mixture after the donor activation to shift the equilibrium
from oxazoline 24 to the oxazolinium ion 22 (Scheme 6b).
Following this third-generation procedure, although the
reaction of 11 and 12 was not affected, the yields for 25 with
13 and 28 were enhanced to 60% and 40%, respectively
(Table 1, entry 3). The acid-sensitive benzylidene moieties
were found to be stable under these conditions. To further
improve on the glycosylation, Lewis acid trimethylsilyl tri-
flate (TMSOTf) was examined as an alternative to TfOH.
Based on this protocol, significant enhancements were ob-
served for all three reactions (Table 1, entry 4), with the yield
of decasaccharide 10 reaching 77%, which enabled us to
acquire over 200 mg of this decasaccharide. These results
suggest that the addition of TMSOTf successfully sup-
pressed the formation of trichlorooxazoline byproduct.
Deprotection of large complex oligosaccharides could be
very challenging due to the presence of multiple protective
groups, as observed in our attempts on decasaccharides 4
and 8. To deprotect decasaccharide 10, its TBS group was
removed first by hydrogen fluoride (HF) in pyridine to give
29 in 79% yield. This was followed by a mild basic condition
(34) Donohoe, T. J.; Logan, J. G.; Laffan, D. D. P. Org. Lett. 2003, 5,
4995–4998.
(35) Sherman, A. A.; Yudina, O. N.; Mironov, Y. V.; Sukhova, E. V.;
Shashkov, A. S.; Menshov, V. M.; Nifantiev, N. E. Carbohydr. Res. 2001,
336, 13–46.
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TABLE 1. Effects of Additives on Glycosylation Product Distribution
entries
1
reaction conditions
donor 11 þ acceptor 12
20 (100%)
donor 25 þ acceptor 13
donor 25 þ acceptor 28
first generation
TTBP was added after donor was activated
second generation
no TTBP was added after donor was activated
third generation
TfOH was added after donor was activated
fourth generation
TMSOTf was added after donor was activated
2
3
4
25 (55%) þ 20 (40%)
25 (55%)
26 (40%) þ 27 (50%)
26 (60%)
10 (10%) þ 27 (85%)
10 (40%)
25 (82%)
26 (71%)
10 (77%)
SCHEME 7
(20 equivalents of KOH added in 10 portions to a solution of
29 over 2 days20) in order to cleave all ten carboxylic esters
and five trichloroacetamides (Scheme 7). Harsh basic con-
ditions should be avoided to prevent epimerization of the
glucuronic acids. The progress of the hydrolysis turned out
to be difficult to monitor by TLC for two reasons. First, the
liberated amino groups gave no ninhydrin test presumably
due to steric hindrance. Second, the compounds with free
carboxylic acid and amino groups became zwitterionic,
which streaked on TLC. Mass spectrometry analysis of the
crude reaction mixture did not yield much information
resulting from signal suppression by the presence of salts.
NMR analysis of the reaction mixture turned out to be most
removal of all five Phth was found to be incompatible with
the carboxylic esters present in the decasaccharide, TCA
could be removed together with the esters under a mild basic
condition. The TCA-associated oxazoline formation side
reaction during glycosylation was suppressed by addition
of TMSOTf to the reaction mixture. Our experience high-
lights the challenges associated with assembly of large oli-
gosaccharides, as the conditions employed for synthesis of
shorter counterparts may not be directly translatable and
each synthesis needs individual optimization and develop-
ment. We believe our strategy on the HA decasaccharide
assembly can open up possibilities to chemical synthesis of
longer HA oligomers and facilitate the structure-activity
relationship studies of this important class of molecules.
1
informative. The cluster of peaks around 8.0 ppm in H
NMR assigned to the ortho-hydrogens of benzoyl groups
merged into a doublet-like pattern over time signifying the
cleavage of benzoyl esters to benzoate salt. Another char-
acteristic of the NMR is that the set of four doublets around
7.0 ppm, representing the amide protons of trichloroacet-
amide groups in 29, became smaller and eventually disap-
peared as the reaction proceeded. The rate of trichloro-
acetamide cleavage was much slower compared with that
of the benzoates. When the crude NMR spectra did not show
much change after week 5, the reaction was stopped. The
reaction mixture was N-acetylated followed by hydrogena-
tion and size exclusion chromatography to produce the
fully deprotected HA decasaccharide 9 in 35% yield from
29. NMR analysis indicated all the glycosyl linkages were
β, confirming no epimerizations occurred despite the
prolonged reaction time.
Experimental Section
General Procedure for Deprotection of p-Methoxybenzyl
(PMB) Group. PMB-protected compound (1 mmol) and DDQ
(1.3 equiv) were dissolved in a solvent mixture of dichloro-
methane (CH2Cl2) and a saturated aqueous solution of NaH-
CO3 (v/v 19:1), which was kept in dark. The reaction mixture
was stirred for 2 h from 0 °C to room temperature. A second
portion of DDQ (0.5 equiv) was added to the reaction mixture,
and a third portion of DDQ (0.5 equiv) could be added 15 min
after the addition of the second portion to further push the
reaction to completion. When most starting material disap-
peared, the reaction mixture was diluted with CH2Cl2 and
washed repeatedly with a saturated aqueous solution of NaH-
CO3 until the organic layer become colorless. The organic layer
was concentrated, and the resultant residue was purified by silica
gel chromatography to give the desired compound.
General Procedure for Oxidation of Alcohols to Carboxylic
˚
Acids. After a mixture of alcohol (1 mmol) and MS-4 A (5 g) in
Conclusions
anhydrous DMF was stirred at room temperature for 2 h, a solution
of PDC (6 mmol) in DMF was added dropwise to the reaction. The
reaction mixture was stirred at room temperature until TLC indi-
cated completion of the reaction, which typically required overnight.
The reaction was diluted with ethyl acetate and filtered to remove the
insoluble PDC. The filtrate was washed with brine to remove DMF.
The crude product was then purified by silica gel chromatography
with ethyl acetate/CH2Cl2/methanol solvent systems.
We report here the first chemical synthesis of a fully
deprotected HA decasaccharide. Our preactivation-based
thioglycoside strategy with p-TolSCl/AgOTf as the promo-
ter was successfully applied for efficient construction of the
decasaccharide backbones. However, considerable difficul-
ties were encountered in deprotection, when the selection of
nitrogen protective group turned out to be crucial. While
7612 J. Org. Chem. Vol. 74, No. 20, 2009

Lu et al.
General Procedure for Benzyl Ester Formation. The car-
boxylic acid containing compound was dissolved in CH2Cl2
(5 mL) and treated with phenyldiazomethane solution in diethyl
ether (∼2 equiv)21,36 for 2-3 h until the disappearance of all the
starting material. The crude product was purified by silica gel
chromatography.
JOCFeatured Article
poured onto crushed ice in saturated NaHCO3 (600 mL). The
organic phase was separated and washed again with saturated
NaHCO3 until the pH reached 7, which was then dried over
Na2SO4, filtered, and concentrated. The resulting crude residue
was mixed with Ag2CO3 (2.76 g, 10 mmol), CaSO4 (2.72 g,
20 mmol), and anhydrous methanol (100 mL). The mixture was
stirred at room temperature under N2 protection for 1 day until
TLC showed one major product, on which all three acetyl groups
has already been cleaved because of the basic reaction condition.
The mixture was concentrated and purified by silica column with
CH2Cl2/ MeOH system. Methyl 2-deoxy-2-trichloroacetamido-
β-D-glucopyranoside (18) was afforded (2.70 g, 8.0 mmol, 80%
for two steps). Compound 18 (2.70 g, 8.0 mmol) was mixed
with camphorsulfonic acid (0.84 g, 3.6 mmol) and benzalde-
hyde dimethyl acetal (1.35 mL, 9.6 mmol) in anhydrous toluene
(200 mL). The reaction mixture was stirred under N2 protection
at 80 °C for 1 h. When TLC showed around 70% conversion of
starting material to product, the reaction flask was placed on a
high vacuum rotary evaporator to further push the reaction to
completion. When toluene was completely removed by the high
vacuum rotary evaporator, the reaction mixture was neutralized
with triethylamine in CH2Cl2. The mixture was diluted with
CH2Cl2 (300 mL) and washed with saturated NaHCO3, dried
over Na2SO4, filtered, and concentrated. Recrystallization af-
forded 15 as a white solid (2.39 g, 5.6 mmol 70%): [R]20D -29.3
General Procedure for Removal of tert-Butyldimethylsilyl
(TBS) Groups. The TBS-protected compound (0.5 mmol) was
dissolved in pyridine (4 mL) in a plastic flask followed by the
addition of 65-70% HF pyridine solution (2 mL) at 0 °C. The
3
solution was stirred for 24 h until TLC indicated completion of
the reaction. The reaction mixture was diluted with ethyl acetate
(50 mL) and washed with 10% aqueous CuSO4 solution
(20 mL). The aqueous phase was extracted with ethyl acetate
(30 mL) twice, and the combined organic layers were washed
with a saturated aqueous solution of NaHCO3 to remove HF.
The crude product was purified by silica gel chromatography.
p-Tolyl 4,6-O-Benzylidene-2-deoxy-2-trichloroacetamido-β-D-
glucopyranoside (14). After D-glucosamine hydrochloride (10.78 g,
50 mmol) and sodium methoxide (50 mL 1 M NaOMe/ MeOH,
50 mmol) in methanol were stirred at room temperature under N2
protection for 45 min, trichloroacetyl chloride (5.61 mL, 50 mmol)
and triethylamine (6.95 mL, 50 mmol) were added. After overnight
when ESI-MS indicated that D-glucosamine hydrochloride has
completely disappeared, methanol was removed by a rotary eva-
porator and the flask was placed under high vacuum for 2 h. Acetic
anhydride and pyridine (150 mL, v/v 1:2) were then added, and the
reaction mixture was stirred for 2 days. The mixture was concen-
trated, and the resulting residue was diluted with ethyl acetate and
washed with a saturated aqueous solution of NaHCO3, 10% HCl,
water, and brine. The organic phase was dried over Na2SO4, filtered,
and concentrated. After recrystallization, 1,3,4,6-tetra-O-acetyl-2-
deoxy-2-trichloroacetamido-D-glucopyranoside (16) was obtained
as a white solid (16.26 g, 33 mmol, 66% for two steps). Boron
trifluoride dimethyl etherate (7.75 mL, 89 mmol) was added to a
solution of 16 (4.93 g, 10 mmol) and p-toluenethiol (1.99 g, 16 mmol)
in dry CH2Cl2 (200 mL). The reaction mixture was stirred at room
temperature under N2 protection overnight, diluted with CH2Cl2
(300 mL), washed with saturated NaHCO3, dried over Na2SO4,
filtered, and concentrated. Recrystallization afforded p-tolyl 3,4,6-
tri-O-acetyl-2-deoxy-2-trichloroacetamido-1-thio-β-D-glucopyrano-
side (17) as a white solid (5.01 g, 9 mmol, 90%). Compound 17
(5.01 g, 9.0 mmol) was dissolved in a mixed solvent of CH2Cl2 and
methanol (v/v = 1/1, 200 mL). Sodium methoxide (2.7 mL 1 M
NaOMe/ MeOH, 2.7 mmol) in methanol was added, and the
mixture was stirred at room temperature under N2 protection for
2 h, neutralized with Amberlite IR-120, concentrated, and dried
under vacuum. Benzaldehyde dimethyl acetal (1.52 mL, 10.8 mmol)
was added to a solution of the resulting residue and camphorsulfonic
acid (0.94 g, 4.05 mmol) in anhydrous toluene (200 mL). The
reaction mixture was stirred under N2 protection at 80 °C for 1 h.
When TLC showed around 70% conversion of starting material to
product, the reaction flask was placed on a high vacuum rotary
evaporator to further push the reaction to completion. When toluene
was completely removed by the high vacuum rotary evaporator, the
reaction mixture was neutralized with triethylamine in CH2Cl2. The
mixture was diluted with CH2Cl2 (300 mL) and washed with
saturated NaHCO3, dried over Na2SO4, filtered, and concentrated.
Recrystallization afforded 14 as a white solid (3.27 g, 6.3 mmol, 70%
for two steps), the structure of which was confirmed by comparison
of NMR spectra.21
1
(c 0.5, CH2Cl2); H NMR (500 MHz, CDCl3) δ 3.46-3.59 (m,
6 H, -OCH3, H-2, H-4, H-5), 3.80-3.84 (m, 1 H, H-6),
4.38-4.42 (m, 2 H, H-3, H-6), 4.87 (d, 1 H, J = 8.0 Hz, H-1),
5.57 (s, 1 H, CHPh), 6.93 (d, 1 H, J = 7.0 Hz, NH), 7.38-7.51 (m,
5 H, CHarom); 13C NMR (125 MHz, CDCl3) δ 57.8 (-OCH3),
59.8 (C-2), 66,4 (C-5), 68,8 (C-6), 69.8 (C-3), 81.8 (C-4), 92.5
(CCl3), 101,2 (C-1), 102,2 (CHPh), 126.5-129.6 (Carom), 137.12
(Cq Carom), 162.6 (CdO TCA); HRMS [M þ Na]þ m/z calcd for
C16H18Cl3NNaO6 448.0097, found 448.0088.
p-Tolyl O-(2-O-Benzoyl-3-O-benzyl-4-O-tert-butyldimethylsi-
lyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl)-(1f3)-4,6-O-ben-
zylidene-2-deoxy-1-thio-2-trichloroacetamido-β-D-glucopyrano-
side (19). The mixture of donor 1 (715 mg, 1 mmol) and freshly
˚
activated MS-4 A (800 mg) in diethyl ether (20 mL) was stirred
in a dry ice-2-propanol bath for 1 h, followed by the addition
of AgOTf (771 mg, 3 mmol) in diethyl ether (10 mL). After
5 min, orange-colored p-TolSCl (157 μL, 1 mmol) was added
via a microsyringe. Since the reaction temperature was lower
than the freezing point of p-TolSCl, p-TolSCl was added
directly into the reaction mixture to prevent it from freezing
on the flask wall. The characteristic yellow color of p-TolSCl in
the reaction solution dissipated rapidly within a few seconds,
indicating depletion of p-TolSCl. The temperature at which
preactivation was performed was not crucial as long as it did
not exceed -50 °C. After the donor was completely consumed
according to TLC analysis (about 5 min at -78 °C), a solution
of acceptor 14 (467 mg, 0.9 mmol) and TTBP (248 mg, 1 mmol)
in a mixture solvent of CH2Cl2 and acetonitrile (v/v 19:1, 3 mL)
was then added dropwise within 10 min to the reaction mixture
(acetonitrile was necessary as acceptors 14 were not freely
soluble in CH2Cl2). The reaction mixture was warmed to
-10 °C under stirring over 2 h. Triethylamine (0.5 mL) was
then added, and the mixture was diluted with CH2Cl2 (100 mL)
and filtered over Celite. The Celite was further washed with
CH2Cl2 until no organic compounds were observed in the
filtrate by TLC. All CH2Cl2 solutions were combined and
washed twice with a saturated aqueous solution of NaHCO3
(100 mL) and twice with water (100 mL). The organic layer was
concentrated, and the crude product was purified by silica gel
chromatography. Typical conditions for chromatography are
silica gel/product ∼2000 by weight with a flow rate of approx-
imate 5 mL/min and a solvent gradient of Rf ∼0.1-0.3 in the
ethyl acetate/CH2Cl2/hexanes solvent systems. Compound 19
Methyl 4,6-O-Benzylidene-2-deoxy-2-trichloroacetamido-β-D-
glucopyranoside (15). Compound 16 (4.93 g, 10 mmol) was
dissolved in HBr in acetic acid (30 mL, 33% w/w, 173.6 mmol).
After 6 h, the mixture was diluted with CH2Cl2 (240 mL) and
(36) Huang, L.; Teumelsan, N.; Huang, X. Chem.;Eur. J. 2006, 12,
5246–5252.
J. Org. Chem. Vol. 74, No. 20, 2009 7613

Lu et al.
JOCFeatured Article
(800 mg, 0.72 mmol, 80%) was afforded as a white solid: [R]20
CHarom), 7.30-7.45 (m, 14 H, CHarom), 7.54-7.57 (m, 1 H,
CHarom), 7.92-7.94 (m, 2 H, CHarom Bz); 13C NMR (125 MHz,
CDCl3) δ 21.4 (PhCH3), 57.4 (C-2), 68.1 (COOCH2Ph), 68.7
(C-6), 70.7 (C-5), 72.2 (C-40), 73.2 (C-20), 74.0 (OCH2Ph), 74.7
(C-3), 76.9 (C-50), 80.1 (C-4), 81.0 (C-30), 84.4 (C-1), 92.1 (CCl3),
99.6 (C-1’), 101.4 (CHPh), 126.3-130.2 (CHarom), 133.6, 134.1,
134.8, 137.3, 137.8, 139.2 (Cq STol, Cq CHPh, Cq OBn, Cq Bz, Cq
COOBn), 161.8, 165.3, 169.3 (CdO TCA, CdO COOBn, CdO
Bz); HRMS [M þ Na]þ m/z calcd for C49H46Cl3NNaO12S
1000.1699, found 1000.1706.
D
þ22.2 (c 0.5, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ -0.08 (s,
3 H, SiCH3), -0.05 (s, 3 H, SiCH3), 0.79 (s, 9 H, SiC(CH3)3),
2.35 (s, 3 H, PhCH3), 3.35-3.43 (m, 3 H, H-50, H-60, H-2), 3.49
(ddd, 1 H, J = 4.5 Hz, 5.0 Hz, 9.0 Hz, H-5), 3.56-3.68 (m, 4 H,
H-30, H-40, H-6, H-60), 3.72 (t, 1 H, J = 9.0 Hz, H-4), 3.80 (s, 3
H, CH3 PMB), 4.27 (dd, 1 H, J = 5.0 Hz, 10.5 Hz, H-6), 4.34 (d,
1 H, J = 12.0 Hz, CH2PMP), 4.54 (d, 1 H, J = 11.0 Hz,
CH2Ph), 4.58 (d, 1 H, J = 11.0 Hz, CH2Ph), 4.63 (d, 1 H, J =
12.0 Hz, CH2PMP), 4.66 (t, 1 H, J = 9.0 Hz, H-3), 4.93 (d, 1 H,
J = 8.0 Hz, H-10), 5.20 (t, 1 H, J = 8.0 Hz, H-20), 5.32 (s,
1 H, CHPh), 5.44 (d, 1 H, J = 10.5 Hz, H-1), 6.89 (d, 1 H, J =
8.5 Hz, NH), 7.04-7.11 (m, 7 H, CHarom), 7.20-7.42 (m, 14 H,
CHarom), 7.82-7.84 (m, 2 H, CHarom Bz); 13C NMR (125 MHz,
CDCl3) δ -4.5 (SiCH3), -3.6 (SiCH3), 18.1 (SiC(CH3)3), 21.4
(PhCH3), 26.0 (ꢀ3, SiC(CH3)3), 55.5 (PhOCH3), 57.6 (C-50),
68.7 (C-6), 69.1 (C-60), 70.9 (C-5), 71.7 (C-40), 73.3, (CH2PMP),
74.4 (C-20), 75.0 (CH2Ph), 76.0 (C-3), 77.4 (C-2), 78.8 (C-4),
83.5 (C-30), 85.4 (C-1), 92.5 (CCl3), 98.0 (C-10), 101.3 (CHPh),
114.0 (Carom-2PhOCH3), 126.4-130.2 (CHarom), 133.3, 133.8
(ꢀ2), 137.3, 137.9, 139.0 (Cq SPhCH3, Cq CHPh, Cq Bn, Cq Bz,
Methyl O-(Benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyrano-
syluronate)-(1f3)-4,6-O-benzylidene-2-deoxy-2-trichloroacet-
amido-β-D-glucopyranoside (13). The mixture of donor 1 (715 mg,
1 mmol), acceptor 15 (427 mg, 1 mmol), and freshly activated
˚
MS-4 A (800 mg) in a solvent mixture of CH2Cl2 and acetonitrile
(v/v 40:1, 20 mL) was stirred for 1 h at -78 °C, followed by the
addition of AgOTf (771 mg, 3 mmol) in diethyl ether (10 mL).
After 5 min, p-TolSCl(157μL, 1 mmol)was added viaa syringe to
activate the donor. The yellow color of the reaction disappeared
quickly, and TLC analysis showed the donor was completely
consumed. A solution of TTBP (248 mg, 1 mmol) in dichloro-
methane (1 mL) was then added dropwise to the reaction
mixture. The reaction mixture was warmed to -10 °C under
stirring over 2 h, followed by the same workup and purification
procedures described above for the synthesis of 19. The resulting
disaccharide was subjected to deprotection of PMB, oxidation of
alcohol to carboxylic acid, benzyl ester formation, and TBS
removal reactions following the general procedures to afford
13 (426 mg, 0.48 mmol, 48% for five steps) as a white solid:
[R]20D þ8.2 (c 0.5, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 3.00
(d, 1 H, J = 2.5 Hz, OH), 3.30 (ddd, 1 H, J = 7.0 Hz, 8.0 Hz,
9.5 Hz, H-2), 3.48-3.50 (m, 4 H, -OCH3, H-5), 3.57-3.60 (m,
3 H, H-30, H-50, H-6), 3.76 (t, 1 H, J = 9.0 Hz, H-4), 4.03 (ddd,
1 H, J = 2.5 Hz, 9.0 Hz, 9.0 Hz, H-40), 4.26 (dd, 1 H, J = 5.0 Hz,
10.5 Hz, H-6), 4.59 (dd, 1 H, J = 9.0 Hz, 9.5 Hz, H-3), 4.63 (d,
1 H, J = 11.5 Hz, -CH2Ph), 4.73 (d, 1 H, J =11.5Hz, -CH2Ph),
4.88(d, 1 H, J = 7.5 Hz, H-10), 4.99 (d, 1 H, J = 8.0Hz, H-1), 5.14
(d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.20 (dd, 1 H, J = 7.5 Hz,
9.5 Hz, H-20), 5.26 (d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.36 (s,
1 H, CHPh), 7.05 (d, 1 H, J = 7.0 Hz, NH), 7.08-7.09 (m, 3 H,
CHarom), 7.27-7.40 (m, 14 H, CHarom), 7.54-7.57 (m, 1 H,
CHarom), 7.92-7.94 (m, 2 H, CHarom Bz); 13C NMR (125 MHz,
CDCl3) δ = 57.8 (-OCH3) 59.3 (C-2), 66.3 (C-5), 68.0 (COO-
CH2Ph), 68.8 (C-6), 72.3 (C-40), 73.2 (C-20), 74.0 (C-50), 74.7
(OCH2Ph), 76.2 (C-3), 80.3 (C-4), 81.1 (C-30), 92.1 (CCl3), 99.7
(C-1), 100.0 (C-10), 101.4 (CHPh), 126.2-130.2 (CHarom), 133.5,
134.9, 137.4, 138.0 (Cq CHPh, Cq OBn, Cq Bz, Cq COOBn), 162.3,
165.3, 169.3 (CdO TCA, CdO COOBn, CdO Bz); HRMS
[M þ Na]þ m/z calcd for C43H42Cl3NNaO13 908.1619, found
908.1616.
2-Trichloromethyl O-(Benzyl 2-O-benzoyl-3-O-benzyl-4-O-
tert-butyldimethylsilyl-β-D- glucopyranosyluronate)-(1f3)-4,6-
O-benzylidene-1,2-dideoxy-r-D-glucopyrano-[2,1-d]-2-oxazoline
(20): [R]20D þ54.1 (c 1.0, CH2Cl2); 1H NMR (500 MHz, CDCl3)
δ -0.06 (s, 3 H, SiCH3), -0.05 (s, 3 H, SiCH3), 0.81 (s, 9 H,
SiC(CH3)3), 3.53 (dd, 1 H, J = 5.0 Hz, 10.0 Hz, H-6), 3.61 (t,
1 H, J = 10.0 Hz, H-5), 3.68 (t, 1 H, J = 7.5 Hz, H-30), 3.91 (dd,
1 H, J = 7.5 Hz, 10.0 Hz, H-4), 4.01 (d, 1 H, J = 8.0 Hz, H-50),
4.15-4.18 (m, 2 H, H-3, H-40), 4.22 (dd, 1 H, J = 5.0 Hz, 8.0 Hz,
H-2), 4.34 (dd, 1 H, J = 5.0 Hz, 10.0 Hz, H-6), 4.66 (d, 1 H, J =
12.5 Hz, CH2Ph), 4.69 (d, 1 H, J = 12.5 Hz, CH2Ph), 5.02 (d,
1 H, J = 7.5 Hz, H-10), 5.11 (d, 1 H, J = 12.0 Hz, COOCH2Ph),
5.15 (d, 1 H, J = 12.0 Hz, COOCH2Ph), 5.38 (t, 1 H, J = 7.5 Hz,
H-20), 5.39 (s, 1 H, CHPh), 6.08 (d, 1 H, J = 8.0 Hz, H-1),
7.13-7.17 (m, 4 H, CHarom), 7.31-7.43 (m, 13 H, CHarom),
7.53-7.70 (m, 1 H, CHarom), 8.00-8.10 (m, 2 H, CHarom Bz);
13C NMR (125 MHz, CDCl3): δ = -4.9 (SiCH3), -4.0 (SiCH3),
18.1 (SiC(CH3)3), 26.1 (ꢀ3, SiC(CH3)3), 63.4 (C-5), 67.5
C
arom-4 PhOCH3), 159.6 (Carom-1 PhOCH3), 161.7, 165.3
(CdO TCA, CdO Bz); HRMS [M þ Na]þ m/z calcd for
C56H64Cl3NNaO12SSi 1130.2882, found 1130.2845.
p-Tolyl O-(Benzyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldi-
methylsilyl-β-D-glucopyranosyluronate)-(1f3)-4,6-O-benzylidene-
2-deoxy-1-thio-2-trichloroacetamido-β-D-glucopyranoside (11).
Compound 19 was converted to 11 in three steps (65% for three
steps) following the general procedures for deprotection of PMB,
oxidation of alcohol to carboxylic acid, and benzyl ester forma-
tion: [R]20D þ10.2 (c 0.5, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ
-0.16 (s, 3 H, SiCH3), -0.13 (s, 3 H, SiCH3), 0.74 (s, 9 H,
SiC(CH3)3), 2.36 (s, 3 H, PhCH3), 3.30 (ddd, 1 H, J = 7.5 Hz,
9.0 Hz, 10.5 Hz, H-2), 3.49-3.55 (m, 2 H, H-30, H-5), 3.65-3.70
(m, 2 H, H-6, H-4), 3.83 (d, J = 6.5 Hz, H-50), 4.13 (t, J = 6.5 Hz,
H-40), 4.32 (dd, 1 H, J = 5.0 Hz, 10.0 Hz, H-6), 4.54 (t, 1 H, J =
9.0 Hz, H-3), 4.59 (d, 1 H, J = 11.5 Hz, -CH2Ph), 4.65 (d, 1 H,
J = 11.5 Hz, -CH2Ph), 5.03 (d, 1 H, J = 7.0 Hz, H-10), 5.13 (d,
1 H, J = 10.5 Hz, -COOCH2Ph), 5.17 (d, 1 H, J = 10.5 Hz,
-COOCH2Ph), 5.18 (dd, 1 H, J = 6.0 Hz, 7.0 Hz, H-20), 5.29 (d,
1 H, J = 10.5 Hz, H-1), 5.32 (s, 1 H, CHPh), 6.96 (d, 1 H, J =
7.5 Hz, NH), 7.10-7.18 (m, 7 H, CHarom), 7.26-7.38 (m, 14 H,
CHarom), 7.51-7.54 (m, 1 H, CHarom), 7.88-7.89 (m, 2 H,
CHarom Bz); 13C NMR (125 MHz, CDCl3) δ -5.0 (SiCH3),
-4.3 (SiCH3), 18.0 (SiC(CH3)3), 21.4 (PhCH3), 25.9 (ꢀ3, SiC-
(CH3)3), 57.5 (C-2), 67.4 (COOCH2Ph), 68.8 (C-6), 70.8 (C-5),
71.4 (C-40), 73.7 (OCH2Ph), 74.4 (C-20), 76.5 (C-3), 77.6 (C-50),
80.2 (C-4), 81.3 (C-30), 84.6 (C-1), 92.2 (CCl3), 99.0 (C-10), 101.8
(CHPh), 126.5-130.2 (CHarom), 133.5, 134.2, 135.3, 137.3, 137.9,
139.2 (Cq STol, Cq CHPh, Cq OBn, Cq Bz, Cq COOBn), 161.8,
165.4, 168.6 (CdO TCA, CdO COOBn, CdO Bz); HRMS
[M þ Na]þ calcd for C55H60Cl3NNaO12SSi 1114.2563, found
1114.2560.
p-Tolyl O-(Benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyrano-
syluronate)-(1f3)-4,6-O-benzylidene-2-deoxy-1-thio-2-trichloro-
acetamido-β-D-glucopyranoside (12). Compound 11 was con-
verted to 12 following the general procedure for TBS removal
(90%): [R]20D þ5.5 (c 1.0, CH2Cl2); 1H NMR (500 MHz, CDCl3)
δ 2.36 (s, 3 H, PhCH3), 3.02 (s, 1 H, OH), 3.30 (ddd, 1 H, J =
7.0 Hz, 9.0 Hz, 10.5 Hz,H-2), 3.52 (ddd, 1 H, J = 4.0 Hz, 4.5 Hz,
9.0 Hz, H-5), 3.55-3.60 (m, 3 H, H-30, H-50, H-6), 3.70 (t, 1 H, J =
9.0 Hz, H-4), 4.03 (t, 1 H, J = 9.0 Hz, H-40), 4.27 (dd, 1 H, J =
4.0 Hz, 10.0 Hz, H-6), 4.59 (t, 1 H, J = 9.0 Hz, H-3), 4.62 (d, 1H,
J = 11.5 Hz, -CH2Ph), 4.72 (d, 1 H, J = 11.5 Hz, -CH2Ph),
4.86 (d, 1 H, J = 8.0 Hz, H-10), 5.15 (d, 1 H, J = 11.5 Hz,
-COOCH2Ph), 5.17 (t, 1 H, J = 8.0 Hz, H-20), 5.26 (d, 1 H, J =
11.5 Hz, -COOCH2Ph), 5.32 (s, 1 H, CHPh), 5.37 (d, 1 H, J =
10.5 Hz, H-1), 7.05 (d, 1 H, J = 7.0 Hz, NH), 7.08-7.18 (m, 7 H,
7614 J. Org. Chem. Vol. 74, No. 20, 2009

Lu et al.
JOCFeatured Article
(COOCH2Ph), 68.7 (C-6), 69.0 (C-2), 72.1 (C-4’), 74.1 (C-20),
74.6 (OCH2Ph), 77.4 (C-50), 78.7 (C-4), 80.3 (C-3), 82.3 (C-30),
101.1 (C-10), 101.5 (CHPh), 105.3 (C-1), 110.0 (CCl3), 125.5-
128.7 (CHarom), 133.5, 135.2, 137.1, 137.8 (Cq CHPh, Cq OBn,
Cq Bz, Cq COOBn), 162.5, 165.5, 168.2 (CdN, CdO COOBn,
CdO Bz); HRMS [M þ Na]þ m/z calcd for C48H52Cl3NNaO12-
Si 990.2222, found 990.2231.
(1f3)-O-(4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-
glucopyranosyl)-(1f4)-O-(benzyl 2-O-benzoyl-3-O-benzyl-β-D-
glucopyranosyluronate)-(1f3)-4,6-O-benzylidene-2-deoxy-2-tri-
chloroacetamido-β-D-glucopyranoside (26). After a mixture of
donor 25 (33.4 mg, 17.14 μmol), acceptor 13 (15.2 mg, 17.14
μmol), and MS-AW-300 (200 mg) in a mixture solvent of CH2Cl2
and acetonitrile (v/v 19:1, 2 mL) was stirred at -78 °C for 1 h,
AgOTf (13 mg, 51.4 μmol) in diethyl ether (0.7 mL) was added.
When the temperature of the reaction mixture was reduced to
-78 °C, p-TolSCl (17.14 μmol, 2.46 μL) was added via a micro-
syringe. After 5 min when the yellowish color disappeared,
TMSOTf (0.75 μL, 4.13 μmol) was added via microsyringe. The
reaction mixture was warmed to -10 °C under stirring over 2 h,
followed by the same workup and purification procedures de-
scribed above for the synthesis of 19. Compound 26 (33 mg,
12 μmol, 71%) was afforded as a white solid: [R]20D -1.7 (c 0.5,
CH2Cl2); 1H NMR (500 MHz, CDCl3) δ -0.14 (s, 3 H, SiCH3),
-0.10 (s, 3 H, SiCH3), 0.78 (s, 9 H, SiC(CH3)3), 3.00 (dd, 1 H, J =
4.5 Hz, 9.5 Hz, H-6a/c/e), 3.13 (ddd, 1 H, J = 4.5 Hz, 5.0 Hz,
9.5 Hz, H-5a/c/e), 3.15 (dd, 1 H, J = 4.5 Hz, 9.5 Hz, H-6a/c/e),
p-Tolyl O-(Benzyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldi-
methylsilyl-β-D-glucopyranosyluronate)-(1f3)-O-(4,6-O-benzy-
lidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1f4)-
O-(benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyluronate)-
(1f3)-4,6-O-benzylidene-2-deoxy-1-thio-2-trichloroacetamido-β-
D-glucopyranoside (25). The mixture of donor 11 (37 mg, 33.9
˚
μmol) and freshly activated MS-4 A (300 mg) in diethyl ether
(2.0 mL) was stirred for 1 h at -78 °C, followed by the addition of
AgOTf (26 mg, 101.7 μmol) in diethyl ether (1.0 mL). After 5 min,
p-TolSCl (5.3 μL, 33.9 μmol) was added via a microsyringe to
activate the donor. The characteristic yellow color of p-TolSCl in
the reaction solution dissipated rapidly within a few seconds
indicating depletion of p-TolSCl, and TLC analysis showed the
donor was completely consumed. A solution of acceptor 12
(30 mg, 30.5 μmol) in a mixture solvent of CH2Cl2 and acetonitrile
(v/v 19:1, 0.7 mL) was then added dropwise to the reaction
mixture. After 5 min, TMSOTf (2 μL, 11.0 μmol) was added via
microsyringe. The reaction mixture was warmed to -10 °C under
stirring over 2 h followed by the same workup and purification
procedures described above for the synthesis of 19. Compound 25
(54mg, 27.8 μmol, 82%) was obtained as a whitesolid: [R]20D þ2.9
3.18-3.23 (m, 1 H, H-5a/c/e), 3.42-3.48(m, 3 H, H-5a/c/e, H-6a/c/e
H-2a/c/e), 3.50 (s, 3 H, OCH3), 3.52-3.61 (m, 3 H, H-3a/c/e
,
,
H-3b/d/f x2), 3.61-3.70 (m, 3 H, H-3a/c/e, H-3b/d/f, H-4a/c/e),
3.72-3.78 (m, 2 H, H-2a/c/e x2), 3.81 (dd, 1 H, J = 9.0 Hz,
9.5 Hz, H-4a/c), 3.81 (dd, 1 H, J = 9.0 Hz, 9.5 Hz, H-4a/c), 3.85 (d,
1 H, J = 6.5 Hz, H-5b/d/f), 3.86 (d, 1 H, J = 8.5 Hz, H-5b/d/f), 3.90
(d, 1 H, J = 8.5 Hz, H-5b/d/f), 4.02 (dd, 1 H, J = 4.5 Hz, 10.0 Hz,
H-6a/c/e), 4.04 (dd, 1 H, J = 4.5 Hz, 10.5 Hz, H-6a/c/e), 4.17 (t, 1 H,
J = 6.5 Hz, H-4b/d/f), 4.19 (t, 1 H, J = 8.5 Hz, H-4b/d/f), 4.25 (t,
1 H, J = 8.5 Hz, H-4b/d/f), 4.29 (dd, 1 H, J = 5.5 Hz, 10.5 Hz,
H-6a/c/e), 4.47 (dd, 1 H, J = 9.0 Hz, 9.5 Hz, H-3a/c/e), 4.52 (d, 1 H,
J = 8.0 Hz, H-1c/e), 4.56 (d, 1 H, J = 11.5 Hz, -CH2Ph), 4.57 (d,
1 H, J = 12.0 Hz, -CH2Ph), 4.62 (d, 1 H, J =11.5Hz, -CH2Ph),
4.68 (d, 1 H, J = 12.0 Hz, -CH2Ph), 4.72 (d, 1 H, J = 8.0 Hz,
H-1c/e), 4.75 (d, 1 H, J = 11.5 Hz, -CH2Ph), 4.78 (d, 1 H, J =
11.5 Hz, -CH2Ph), 4.84 (d, 1 H, J = 8.5 Hz, H-1a),4.94 (d, 1 H,
J = 6.0 Hz, H-1b/d/f), 4.97 (d, 1 H, J = 6.5 Hz, H-1b/d/f), 4.99 (d,
1 H, J = 7.0 Hz, H-1b/d/f), 5.01 (d, 1 H, J = 12.0 Hz, -COO-
CH2Ph), 5.04 (d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.07 (dd, 1 H,
J = 6.0 Hz, 8.0 Hz, H-2b/d/f), 5.11 (d, 1 H, J = 12.0 Hz,
-COOCH2Ph), 5.12 (dd, 1 H, J = 6.5 Hz, 8.5 Hz, H-2b/d/f),
5.14 (s, 1 H, CHPh), 5.18 (d, 1 H, J = 12.0 Hz, -COOCH2Ph),
5.19 (s, 1 H, CHPh), 5.20 (d, 1 H, J = 12.0 Hz, -COOCH2Ph),
5.21 (d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.21 (dd, 1 H, J =
6.0 Hz, 7.0 Hz, H-2b/d/f), 5.32 (s, 1 H, CHPh), 6.48 (d, 1 H, J =
8.5 Hz, NH), 6.60 (d, 1 H, J = 8.5 Hz, NH), 6.99 (d, 1 H, J =
8.0 Hz, NH), 7.13-7.60 (m, 54 H, CHarom), 7.89-7.98 (m, 6 H,
CHarom Bz); 13C NMR (125 MHz, CDCl3) δ -5.0 (SiCH3), -4.2
1
(c 0.5, CH2Cl2); H NMR (500 MHz, CDCl3) δ -0.14 (s, 3 H,
SiCH3), -0.11 (s, 3 H, SiCH3), 0.78 (s, 9 H, SiC(CH3)3), 2.31 (s,
3 H, PhCH3), 3.09 (ddd, 1 H, J = 5.0 Hz, 5.0 Hz, 10.0 Hz, H-500),
3.17-3.22 (m, 1 H, H-600), 3.44-3.50 (m, 2 H, H-2, H-5), 3.53 (dd,
1 H, J = 9.5 Hz, 10.0 Hz, H-3), 3.55 (t, 1 H, J = 7.0 Hz, H-3000),
3.58-3.61 (m, 2 H, H-400, H-6), 3.64 (t, 1 H, J=8.0 Hz, H-30), 3.72
(t, 1 H, J = 9.0 Hz, H-200), 3.81 (dd, 1 H, J = 9.0 Hz, 9.5 Hz H-4),
3.86 (d, 1 H, J = 7.0 Hz, H-5000), 3.87 (d, 1 H, J = 8.0 Hz, H-50),
4.02 (dd, 1 H, J = 5.0 Hz, 10.5 Hz, H-600), 4.17 (t, 1 H, J = 7.0 Hz,
H-4000), 4.20 (t, 1 H, J = 8.0 Hz, H-40), 4.29 (dd, 1 H, J = 5.0 Hz,
10.5 Hz, H-6), 4.41 (t, 1 H, J = 9.0 Hz, H-300), 4.53 (d, 1 H, J =
11.5 Hz, -CH2Ph), 4.61 (d, 1 H, J = 9.0 Hz, H-100), 4.61 (d, 1 H,
J = 11.5Hz, -CH2Ph), 4.68 (d, 1 H, J = 11.5 Hz, -CH2Ph), 4.74
(d, 1 H, J = 11.5 Hz, -CH2Ph), 4.97 (d, 1 H, J = 10.0 Hz,
-COOCH2Ph), 5.00 (s, 1 H, CHPh), 5.00 (d, 1 H, J = 7.0 Hz,
H-1000), 5.06 (t, 1 H, J = 8.0 Hz, H-20), 5.12 (d, 1 H, J = 7.5 Hz,
H-1), 5.12 (d, 1 H, J = 10.0 Hz, -COOCH2Ph), 5.16 (d, 1 H, J =
10.0 Hz, -COOCH2Ph), 5.17 (d, 1 H, J = 8.0 Hz, H-10), 5.19 (d,
1 H, J = 11.5Hz, -COOCH2Ph), 5.20 (t, 1 H, J = 7.0 Hz, H-2000),
5.32 (s, 1 H, CHPh), 6.49 (d, 1 H, J = 9.0 Hz, NH), 7.08 (d, 1 H,
J = 7.5 Hz, NH00), 7.10-7.20 (m, 13 H, CHarom), 7.26-7.42 (m,
25 H, CHarom), 7.53-7.58 (m, 2 H, CHarom), 7.89-7.91 (m, 2 H,
CHarom Bz), 7.95-7.97 (m, 2 H, CHarom Bz); 13C NMR (125
MHz, CDCl3) δ -5.0 (SiCH3), -4.2 (SiCH3), 18.1 (SiC(CH3)3),
21.4 (PhCH3), 26.0 (ꢀ3, SiC(CH3)3), 56.8 (C-2), 57.6 (C-200), 66.4
(C-500), 67.4 (COOCH2Ph), 68.4 (COOCH2Ph),68.0 (C-6), 68.0
(C-600), 70.8 (C-5), 71.4 (C-4000), 73.7 (OCH2Ph), 74.6 (OCH2Ph),
74.6 (C-2000), 75.0 (C-20), 75.0 (C-50), 75.8 (C-4), 77.0 (C-300), 77.3
(C-40), 77.8 (C-5000), 78.5 (C-30), 79.8 (C-400), 80.2 (C-3), 81.4 (C-
3000), 85.6 (C-1), 92.4 (CCl3), 92.7 (CCl3), 99.3 (CHPh), 100.0,
100.0 (C-100, C-1000), 101.0 (CHPh), 101.7 (C-10), 126.2-130.1
(CHarom), 133.5, 133.5, 134.1, 135.4, 135.4, 137.4, 137.4, 138.0,
138.4, 138.4 (Cq STol, Cq CHPh ꢀ 2, Cq OBn ꢀ 2, Cq Bz ꢀ 2,
Cq COOBn ꢀ 2), 161.7, 165.0, 165.3, 165.7, 168.5, 169.1 (CdO
TCA ꢀ 2, CdO COOBn ꢀ 2, CdO Bz ꢀ 2); HRMS [M þ H]þ
m/z calcd for C97H99Cl6N2O24SSi 1949.4191, found 1949.4142.
Methyl O-(Benzyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldi-
methylsilyl-β-D-glucopyranosyluronate)-(1f3)-O-(4,6-O-benzy-
lidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1f4)-
O-(benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyluronate)-
(SiCH3), 18.1 (SiC(C ) ), 26.0 (ꢀ3, SiC(CH3)3), 57.2 (C-2a/c/e),
3 3
57.5 (C-2a/c/e), 57.7 (OCH3),58.8(C-2a/c/e), 66.3, 66.4, 66.4 (C-5a/c/e
,
C-6a/c/e, C-5a/c/e), 67.4 (COOCH2Ph), 68.0 (COOCH2Ph), 68.1
(COOCH2Ph), 68.3, 68.6, 68.7 (C-6a/c/e, C-6a/c/e, C-5a/c/e), 71.4
(C-4f), 73.7 (OCH2Ph), 74.3 (C-2b/d/f), 74.7 (OCH2Ph), 74.7
(OCH2Ph), 74.7 (ꢀ2, C-5b/d/f x2), 75.0 (C-2b/d/f), 75.1 (C-2b/d/f),
76.0 (C-3a/c/e), 76.8 (C-4a/c/e), 77.0 (C-4a/c/e), 77.5 (C-4b/d), 77.6
(C-4b/d), 77.8 (C-5b/d/f), 78.8 (C-3b/d/f), 79.0 (C-3b/d/f), 80.0 (ꢀ2,
C-3a/c/e ꢀ 2), 82.2 (C-4a/c/e), 81.5(C-3b/d/f), 92.4 (CCl3), 92.6 (CCl3),
92.7 (CCl3), 99.3 (C-1b/d/f), 99.9 (C-1b/d/f), 100.0 (C-1c/e), 100.1
(C-1c/e), 100.3 (C-1b/d/f), 100.8 (C-1a), 100.9 (CHPh), 101.1
(CHPh), 101.7 (CHPh), 126.3-130.4 (CHarom), 133.4, 133.5,
133.6, 135.0 (ꢀ2), 135.4, 137.4, 137.5, 137.5, 137.9, 138.3, 138.5
(Cq CHPh ꢀ 3, Cq OBn ꢀ 3, Cq Bz ꢀ 3, Cq COOBn ꢀ 3), 161.6,
162.0, 162.3, 165.3, 165.6, 165.6, 168.6, 169.0, 169.1 (CdO TCA ꢀ
3, CdO COOBn ꢀ 3, CdO Bz ꢀ 3); HRMS [M þ Na]þ m/z calcd
for C133H132Cl9N3NaO37Si 2734.5383, found 2734.5469.
2-Trichloromethyl O-(Benzyl 2-O-benzoyl-3-O-benzyl-4-O-
tert-butyldimethylsilyl-β-D-glucopyranosyluronate)-(1f3)-O-(4,6-
O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-
J. Org. Chem. Vol. 74, No. 20, 2009 7615

Lu et al.
JOCFeatured Article
(1f4)-O-(benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyl-
uronate)-(1f3)-4,6-O-benzylidene-1,2-dideoxy-r-D-glucopyrano-
[2,1-d]-2-oxazoline (27): [R]20D þ19.3 (c 0.5, CH2Cl2); 1H NMR
(500 MHz, CDCl3) δ -0.15 (s, 3 H, SiCH3), -0.12 (s, 3 H,
SiCH3), 0.77 (s, 9 H, SiC(CH3)3), 3.02 (ddd, 1 H, J = 4.5 Hz,
5.0 Hz, 9.5 Hz, H-500), 3.26-3.31 (m, 1 H, H-600), 3.44-3.48 (m,
2 H, H-6, H-5), 3.55 (t, 1 H, J = 7.0 Hz, H-3000), 3.63 (t, 1 H, J =
9.5 Hz, H-400), 3.73-3.68(m, 2 H, H-30, H-200), 3.82-3.91 (m, 4 H,
H-3, H-4, H-50, H-5000), 4.04 (dd, 1 H, J = 5.0 Hz, 11.0 Hz, H-600),
4.13-4.23 (m, 4 H, H-2, H-300, H-40, H-4000), 4.28-4.29 (m, 1 H,
H-6), 4.55 (d, 1 H, J = 8.0 Hz, H-100), 4.57 (d, 1 H, J = 11.5 Hz,
-CH2Ph), 4.60 (d, 1 H, J = 11.5 Hz, -CH2Ph), 4.67 (d, 1 H, J =
11.5 Hz, -CH2Ph), 4.77 (d, 1 H, J = 11.5 Hz, -CH2Ph), 4.89 (d,
1H, J=7.5Hz, H-1000), 4.95(d, 1H, J=12.0Hz, -COOCH2Ph),
5.02 (d, 1 H, J = 7.0 Hz, H-10), 5.15 (d, 1 H, J = 12.0 Hz, -COO-
CH2Ph), 5.17 (s, 1 H, CHPh), 5.19 (d, 1 H, J = 12.0 Hz, -COO-
CH2Ph), 5.20 (d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.20 (t, 1 H,
J = 7.0 Hz, H-20), 5.24 (dd, 1 H, J = 7.5 Hz, 8.5 Hz, H-2000), 5.34
(s, 1 H, CHPh), 6.09 (d, 1 H, J = 7.5 Hz, H-1), 6.58 (d, 1 H, J =
8.5 Hz, NH00), 7.10-7.18 (m, 10 H, CHarom), 7.29-7.45 (m, 22 H,
CHarom), 7.59-7.52 (m, 2 H, CHarom), 7.95-8.00 (m, 4 H, CHarom
Bz); 13C NMR (125 MHz, CDCl3) δ -5.0 (SiCH3), -4.2 (SiCH3),
18.1 (SiC(CH3)3), 26.0 (ꢀ3, SiC(CH3)3), 57.6 (C-200), 63.4 (C-5),
66.4 (C-500), 67.4 (COOCH2Ph), 67.8 (COOCH2Ph), 68.6 (C-6),
68.6 (C-600), 68.9 (C-2), 71.4 (C-4000), 73.1 (C-2000), 73.7 (OCH2Ph),
74.7 (C-20), 74.8 (OCH2Ph), 75.2 (C-5), 76.7 (C-400), 77.1 (C-300),
77.8 (C-5000), 78.5 (C-4), 79.9 (C-30), 80.1 (C-40), 80.2 (C-3), 81.4
(C-3000), 92.6 (CCl3, TCA), 99.2 (C-10), 99.4 (C-100), 101.0 (C-1000),
101.3 (CHPh), 101.7 (CHPh), 110.0 (NdC-CCl3), 105.2 (C-1),
126.2-130.1 (CHarom), 133.4 ꢀ 2, 135.1, 135.4, 137.1, 137.4, 138.0,
138.2 (Cq CHPh ꢀ 2, Cq OBn ꢀ 2, Cq Bz ꢀ 2, Cq COOBn ꢀ 2),
161.8, 162.5, 165.3, 165.4, 168.2, 168.5 (CdN-CCl3, CdO TCA,
CdOCOOBnꢀ 2, CdOBzꢀ 2);HRMS[Mþ Na]þ m/zcalcd for
C90H90Cl6N2NaO24Si 1843.3682, found 1843.3724.
8.5 Hz, NH), 6.63 (d, 1 H, J= 9.0 Hz, NH), 7.02 (d, 1 H, J=7.5Hz,
NH), 7.13-7.61(m,54H,CHarom), 7.90-8.04 (m, 6 H, CHarom Bz);
13C NMR (125 MHz, CDCl3) δ 57.1 (C-2a/c/e), 57.5 (C-2a/c/e), 57.7
,
(OCH3), 58.8 (C-2a/c/e), 66.3, 66.4 (C-5a/c/e C-6a/c/e), 66.4
(COOCH2Ph), 68.0 (COOCH2Ph), 68.1 (COOCH2Ph), 68.1, 68.3,
68.5, 68.7 (C-6a/c/e ꢀ 2, C-5a/c/e ꢀ 2), 72.0 (C-4f), 73.6 (C-2b/d/f), 74.2
(C-2b/d/f), 74.3 (C-5b/d), 74.6 (OCH2Ph), 74.8 (OCH2Ph), 74.7
(OCH2Ph), 74.9 (C-5b/d), 75.2 (C-2b/d/f), 76.0 (C-3f), 76.9 (C-4a/c/e),
77.2 (C-4b/d), 77.4 (C-4b/d), 77.6 (C-5f), 78.8 (C-3a/c/e), 79.0 (C-3a/c/e),
80.1, 80.1, 80.1 ꢀ 2 (C-3b/d/f ꢀ 2, C-4a/c/e ꢀ 2), 81.1 (C-3a/c/e), 92.4
(CCl3), 92.6 (CCl3), 92.7 (CCl3), 99.8 (C-1b/d/f), 99.9 (C-1b/d/f), 100.0
(C-1c/e), 100.0 (C-1c/e), 100.4 (C-1b/d/f), 100.8 (C-1a), 100.9 (CHPh),
101.1 (CHPh), 101.4 (CHPh), 126.1-130.3 (CHarom), 133.5, 133.6,
133.6, 134.9, 135.0, 135.1, 137.4, 137.5 ꢀ 2, 138.0, 138.3, 138.5 (Cq
CHPh ꢀ 3, Cq OBn ꢀ 3, Cq Bz ꢀ 3, Cq COOBn ꢀ 3), 161.7, 162.0,
162.4, 165.1, 165.6, 165.7, 169.0, 169.3, 169.3 (CdO TCA ꢀ 3, CdO
COOBn ꢀ 3, CdO Bz ꢀ 3); HRMS [M þ Na]þ m/z calcd for
C127H118Cl9N3NaO37 2620.4513, found 2620.4595.
Methyl O-(Benzyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldi-
methylsilyl-β-D-glucopyranosyluronate)-(1f3)-O-(4,6-O-benzyli-
dene-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1f4)-O-
(benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyluronate)-(1f3)-
O-(4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-gluco-
pyranosyl)-(1f4)-O-(benzyl 2-O-benzoyl-3-O-benzyl-β-D-gluco-
pyranosyluronate)-(1f3)-O-(4,6-O-benzylidene-2-deoxy-2-tri-
chloroacetamido-β-D-glucopyranosyl)-(1f4)-O-(benzyl 2-O-ben-
zoyl-3-O-benzyl-β-D-glucopyranosyluronate)-(1f3)-O-(4,6-O-ben-
zylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1f4)-
O-(benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyluronate)-
(1f3)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-gluco-
pyranoside (10). After a mixture of donor 25 (33.4 mg, 17.14
μmol), acceptor28(25mg, 9.62 μmol), and MS-AW-300 (200 mg)
in a mixture solvent of CH2Cl2 and acetonitrile (v/v 19:1, 2 mL)
was stirred at -78 °C for 1 h, AgOTf (51.4 mg, 20 μmol) in diethyl
ether (0.7 mL) was added. When the temperature of the reaction
mixture reached -78 °C, p-toluenesulfenyl chloride (p-TolSCl)
(17.1 μmol, 2.5 μL) was added via a microsyringe. After 5 min
when the yellowish color disappeared, TMSOTf (1.25 μL, 6.9
μmol) was added via microsyringe. The reaction mixture was
warmed to -10 °C under stirring over 2 h, followed by the same
workup and purification procedures described above for the
synthesis of 19. Compound 10 (32.6 mg, 7.37 μmol, 76.6%) was
afforded as a white solid: [R]20D -2.7 (c 0.5, CH2Cl2); 1H NMR
(500 MHz, CDCl3) δ -0.14 (s, 3 H, SiCH3), -0.10 (s, 3 H,
SiCH3), 0.79 (s, 9 H, SiC(CH3)3), 3.01 (dd, 1 H, J = 5.0 Hz,
9.0 Hz, H-5a/c/e/g/i), 3.03 (dd, 1 H, J = 5.0 Hz, 9.0 Hz, H-5a/c/e/g/i),
3.05 (dd, 1 H, J = 5.0 Hz, 9.0 Hz, H-5a/c/e/g/i), 3.12-3.22 (m,
Methyl O-(Benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyrano-
syluronate)-(1f3)-O-(4,6-O-benzylidene-2-deoxy-2-trichloroace-
tamido-β-D-glucopyranosyl)-(1f4)-O-(benzyl 2-O-benzoyl-3-O-
benzyl-β-D-glucopyranosyluronate)-(1f3)-O-(4,6-O-benzylidene-
2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1f4)-O-(benzyl
2-O-benzoyl-3-O- benzyl-β-D-glucopyranosyluronate)-(1f3)-4,6-O-
benzylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside (28).
Compound 26 was converted to 28 following the general procedure
for TBS removal as described previously (90%): [R]20D -3.0 (c 0.5,
1
CH2Cl2); H NMR (500 MHz, CDCl3) δ 3.00 (ddd, 1 H, J =
5.0 Hz, 5.0 Hz, 10.0 Hz, H-5a/c/e), 3.07 (s, 1 H, OH), 3.12-3.20 (m,
3 H, H-6a/c/e ꢀ 3), 3.43-3.48 (m, 3 H, H-5a/c/e ꢀ 2, H-2a/c/e), 3.50 (s,
3 H, OCH3), 3.61 (t, 2 H, J = 9.0 Hz, H-3a/c/e ꢀ 2), 3.63-3.70 (m,
3 H, H-3 b/d/f ꢀ 2, H-4a/c/e), 3.72-3.79 (m, 2 H, H-2a/c/e ꢀ 2), 3.82
(dd, 1 H, J = 8.5 Hz, 9.0 Hz, H-4a/c/e), 3.82 (dd, 1 H, J = 8.5 Hz,
9.5 Hz, H-4a/c/e), 3.87 (d, 1 H, J = 8.5 Hz, H-5b/d), 3.90 (d, 1 H, J =
8.0 Hz, H-5b/d), 4.02(dd, 1H, J= 4.5 Hz, 9.5 Hz, H-6a/c/e), 4.05(dd,
5 H, H-5a/c/e/g/i, H-6a/c/e/g/i ꢀ 4), 3.42-3.48 (m, 3 H, H-2a/c/e/g/i
,
H-5a/c/e/g/i, H-6a/c/e/g/i), 3.50 (s, 3 H, OCH3), 3.53 (dd, 1 H, J =
7.0 Hz, 7.5 Hz), 3.55 (dd, 1 H, J = 6.0 Hz, 7.0 Hz), 3.56 (dd, 1 H,
J = 5.5 Hz, 6.0 Hz), 3.60 (dd, 2 H, J = 8.5 Hz, 10.0 Hz), 3.66 (dd,
1 H, J = 6.5 Hz, 8.5 Hz), 3.67 (t, 2 H, J = 8.0 Hz), 3.68 (dd, 2 H,
J = 8.5 Hz, 9.0 Hz) (H-4a/c/e/g/i, H-3a/c/e/g/i ꢀ 4, H-3b/d/f/h/j ꢀ 5),
3.72-3.78 (m, 4 H, H-2a/c/e/g/i ꢀ 4), 3.80 (dd, 2 H, J = 9.0 Hz,
9.5 Hz, H-4b/d/f/h/j ꢀ 2), 3.81 (dd, 2 H, J = 9.0 Hz, 9.5 Hz,
H-4b/d/f/h/j ꢀ 2), 3.85 (d, 1 H, J = 6.5 Hz, H-5b/d/f/h/j), 3.86 (d, 1 H,
J = 7.0 Hz, H-5b/d/f/h/j), 3.88 (d, 2 H, J = 9.0 Hz, H-5b/d/f/h/j ꢀ 2),
3.90 (d, 1 H, J = 8.5 Hz, H-5b/d/f/h/j), 4.01-4.06 (m, 4 H,
H-6a/c/e/g/i ꢀ 4), 4.14 (t, 1 H, J = 7.0 Hz, H-4b/d/f/h/j), 4.16 (t,
1 H, J = 7.0 Hz, H-4b/d/f/h/j), 4.18 (t, 1 H, J = 8.5 Hz, H-4b/d/f/h/j),
4.24 (dd, 1 H, J = 8.5 Hz, 9.0 Hz, H-4b/d/f/h/j), 4.25 (dd, 1 H, J =
8.5 Hz, 9.0 Hz, H-4b/d/f/h/j), 4.28 (dd, 1 H, J = 5.0 Hz, 10.5 Hz,
H-6a/c/e/g/i), 4.48 (dd, 1 H, J = 9.0 Hz, 9.5 Hz, H-3a/c/e/g/i), 4.53 (d,
1 H, J = 8.0 Hz, H-1a/c/e/g/i), 4.56 (d, 1 H, J = 11.5 Hz, -CH2Ph),
4.56 (d, 1 H, J = 11.5 Hz, -CH2Ph), 4.56 (d, 1 H, J = 8.5 Hz,
H-1a/c/e/g/i), 4.57 (d, 1 H, J =11.5Hz, -CH2Ph), 4.62 (d, 1 H, J =
11.5 Hz, -CH2Ph), 4.63 (d, 1 H, J = 8.0 Hz, H-1a/c/e/g/i), 4.63 (d,
1 H, J = 11.5 Hz, -CH2Ph), 4.69 (d, 1 H, J =11.5Hz, -CH2Ph),
1 H, J = 4.5 Hz, 11.0 Hz, H-6a/c/e), 4.08-4.18 (m, 3H, H-3b/d/f
,
H-4b/d/f ꢀ 2), 4.19 (dd, 1 H, J = 8.0 Hz, 8.5 Hz, H-4b/d/f), 4.25 (d,
1 H, J = 8.5 Hz, H-5b/d/f), 4.29 (dd, 1 H, J = 5.5 Hz, 10.5 Hz,
H-6a/c/e), 4.48 (dd, 1 H, J = 9.0 Hz, 9.5 Hz, H-3a/c/e), 4.52 (d, 1 H,
J = 8.0 Hz, H-1c/e), 4.56 (d, 1 H, J = 12.0 Hz, -CH2Ph), 4.57 (d,
1 H, J = 12.0 Hz, -CH2Ph), 4.66 (d, 1 H, J = 11.5 Hz, -CH2Ph),
4.75 (d, 1 H, J = 7.5 Hz, H-1c/e), 4.75 (d, 1 H, J = 11.5 Hz,
-CH2Ph), 4.76 (d, 1 H, J = 12.0 Hz, -CH2Ph), 4.79 (d, 1 H, J =
12.0 Hz, -CH2Ph),4.85(d,1H,J=8.0Hz,H-1a),4.85(d,1H,J=
7.5 Hz, H-1b/d/f), 4.94-4.98 (m, 2 H, H-1b/d/f ꢀ 2), 5.02 (d, 1 H, J =
11.5 Hz, -COOCH2Ph), 5.04 (d, 1 H, J=12.0Hz, -COOCH2Ph),
5.06 (dd, 1 H, J = 8.0 Hz, 8.5 Hz, H-2b/d/f), 5.10 (d, 1 H, J =
12.0 Hz, -COOCH2Ph), 5.13 (s, 1 H, CHPh), 5.16 (d, 1 H, J =
12.0 Hz, -COOCH2Ph), 5.16 (d, 1 H, J=12.0Hz, -COOCH2Ph),
5.17 (dd, 1 H, J = 7.5 Hz, 8.5 Hz, H-2b/d/f), 5.19 (dd, 1 H, J =
6.5 Hz, 8.0 Hz, H-2b/d/f), 5.21 (s, 1 H, CHPh), 5.25 (d, 1 H, J =
11.5 Hz, -COOCH2Ph), 5.32 (s, 1 H, CHPh), 6.45 (d, 1H, J =
7616 J. Org. Chem. Vol. 74, No. 20, 2009

Lu et al.
JOCFeatured Article
4.75 (d, 1 H, J = 8.0 Hz, H-1a/c/e/g/i), 4.76 (d, 1 H, J = 11.5 Hz,
-CH2Ph), 4.79 (d, 1 H, J = 11.5 Hz, -CH2Ph x3), 4.83 (d, 1 H,
J = 8.0 Hz, H-1a/c/e/g/i), 4.91 (t, 2 H, J = 6.0 Hz, H-2b/d/f/h/j ꢀ 2),
4.95 (d, 1 H, J = 6.0 Hz, H-1b/d/f/h/j), 4.96 (d, 1 H, J = 6.0 Hz,
H-1b/d/f/h/j), 5.00 (d, 1 H, J = 7.0Hz, H-1b/d/f/h/j), 4.98 (d, 1 H, J =
11.0 Hz, -COOCH2Ph), 5.00 (d, 1 H, J = 12.0 Hz, -COO-
CH2Ph), 5.02 (d, 1 H, J = 11.5 Hz, -COOCH2Ph ꢀ 2), 5.06 (d,
1 H, J = 8.0 Hz, H-1b/d/f/h/j), 5.06 (d, 1 H, J = 8.0 Hz, H-1b/d/f/h/j),
5.06 (d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.09 (dd, 1 H, J =
8.0 Hz, 9.0 Hz, H-2b/d/f/h/j), 5.12 (d, 1 H, J = 11.5 Hz, -COO-
CH2Ph), 5.13 (s, 3 H, CHPh ꢀ 3), 5.15 (d, 1 H, J = 11.5 Hz,
-COOCH2Ph), 5.15 (dd, 1 H, J = 8.0 Hz, 9.0 Hz, H-2b/d/f/h/j),
5.19 (d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.19 (s, 1 H, CHPh),
5.20 (d, 1 H, J = 11.0 Hz, -COOCH2Ph), 5.21 (d, 1 H, J =
12.0 Hz, -COOCH2Ph), 5.21 (dd, 1 H, J = 7.0 Hz, 8.5 Hz,
H-2b/d/f/h/j), 5.31 (s, 1H, CHPh), 6.50 (d, 1 H, J = 9.0 Hz, NH),
6.55 (d, 2 H, J = 8.0 Hz, NH ꢀ 2), 6.64 (d, 1 H, J = 9.0 Hz, NH),
7.02 (d, 1 H, J = 7.5 Hz, NH), 7.10-7.58 (m, 90 H, CHarom),
7.90-7.95 (m, 10 H, CHarom Bz); 13C NMR (125 MHz, CDCl3) δ
previously (79%). Compound 29 (44 mg, 10 μmol) was dissolved
in 2 mL of THF. A 0.2 M KOH aqueous solution (1 mL, 200
μmol) was added in 10 portions during 2 days. The reaction
mixture was stirred at room temperature during which time the
ratio of water to THF was gradually increased for a better
solubility of the reaction mixture. The reaction was checked by
comparisons of the crude 1H NMR spectra which was taken by
the end of each week. The reaction was stopped by the neutra-
lization with 0.2 M acetic acid in water by the end of the fifth
week when peaks around 7.0 ppm in the 1H NMR of the crude
NMR indicating NHTCA groups disappeared and peaks
around 8.0 peaks for ortho-hydrogens on the benzoyl groups
changed from several equally height peaks into two main peaks.
The solvent was completely removed by high vacuum rotary
evaporator, and the crude residue was dissolved in anhydrous
methanol (3 mL) and cooled to 0 °C. Triethylamine (7.0 μL,
50 μmol) and acetic anhydride (118 μL, 1.25 mmol) were added,
and the reaction mixture was stirred for 2 days under N2
protection at room temperature. The reaction was stopped when
TLC (acetic acid/CH2Cl2/methanol solvent systems) showed
formation of one main product. The reaction solvent was
removed by rotary evaporation, and silica column purification
(acetic acid/CH2Cl2/methanol solvent systems) yielded a mix-
ture mainly composed of 31. Pd(OH)2 (50 mg) was added to a
solution of the 31 in THF (0.5 mL), methanol (0.7 mL), and
acetic acid (0.5 mL). The reaction flask was evacuated using a
water aspirator and filled with hydrogen. This process was
repeated three times, and the reaction mixture was stirred under
hydrogen atmosphere for 3 days. The solution was filtered and
concentrated, and the crude product was purified by Sephadex
G-15 size-exclusion chromatography to give the desired product
-5.0 (SiCH3), -4.2 (SiCH3), 18.1 (SiC(C ) ), 26.0 (ꢀ3, SiC-
3 3
(CH3)3), 57.1, 57.1 (ꢀ2) (C-2a/c/e/g/i ꢀ 3), 57.8 (C-2a/c/e/g/i), 57.8
(OCH3), 58.7 (C-2a/c/e/g/i), 66.4, 66.3 (ꢀ2), 66.3 (ꢀ2) (C-5a/c/e/g/i
ꢀ
5), 67.4 (COOCH2Ph), 68.1 (ꢀ5, COOCH2Ph ꢀ 4, C-6a/c/e/g/i),
68.4 (ꢀ2, C-6a/c/e/g/i ꢀ 2), 68.6 (C-6a/c/e/g/i), 68.7 (C-6a/c/e/g/i), 71.4
(C-4j), 73.7 (OCH2Ph), 74.3 (C-2b/d/f/h/j), 74.6 (C-2b/d/f/h/j), 74.7
(OCH2Ph), 74.8 (ꢀ7), 74.9 (OCH2Ph ꢀ 3, C-5b/d/f/h/j ꢀ 5), 75.0
(C-2b/d/f/h/j), 75.0 (C-2b/d/f/h/j), 75.2 (C-2b/d/f/h/j), 76.0 (C-3a/c/e/g/i),
77.8, 77.6, 77.4 (ꢀ 2) (C-4b/d/f/h ꢀ 4), 77.1 (ꢀ3), 76.9 (C-4a/c/e/g/i
ꢀ
4), 81.4, 83.3, 80.1 (ꢀ4), 79.0, 79.0 (ꢀ2), 78.8 (C-3b/d/f/h/j ꢀ 5,
C-3a/c/e/g/i ꢀ 4, C-4a/c/e/g/i), 92.4, 92.6, 92.7, 92.8 (ꢀ2) (CCl3 ꢀ 5),
99.3 (C-1b/d/f/h/j), 100.0 (C-1a/c/e/g/i), 100.0 (C-1a/c/e/g/i), 100.2
(C-1b/d/f/h/j), 100.3 (C-1a/c/e/g/i ꢀ 2), 100.3 (C-1b/d/f/h/j ꢀ 3), 100.8
(C-1a/c/e/g/i), 100.9 (CHPh), 100.9 (CHPh ꢀ 2), 101.1 (CHPh),
101.7 (CHPh), 126.2-130.2 (CHarom), 133.5, 133.5 (ꢀ2), 133.5,
133.6, 135.0 (ꢀ2), 135.0 (x2), 135.4, 137.4, 137.5 (x2), 137.5,
137.5, 138.0, 138.3, 138.5 (x2), 138.5 (Cq CHPh x5, Cq OBn x5, Cq
Bz ꢀ 5, Cq COOBn ꢀ 5), 161.6, 161.9 (ꢀ2), 162.0, 162.3, 165.3,
165.6, 165.6, 165.7, 165.6, 168.6, 169.1 (ꢀ2), 169.3, 169.4 (CdO
TCA ꢀ 5, CdO COOBn ꢀ 5, CdO Bz ꢀ 5); MALDI [M þ Na]þ
m/z calcd for C217H208Cl15N5NaO61Si 4442.85, found 4443.15.
Methyl O-(β-D-Glucopyranosyluronic acid)-(1f3)-O-(2-N-
acetyl-2-deoxy-β-D-glucopyranosyl)-(1f4)-O-(β-D-glucopyrano-
syluronic acid)-(1f3)-O-(2-N-acetyl-2-deoxy-β-D-glucopyranosyl)-
(1f4)-O-(β-D-glucopyranosyluronic acid)-(1f3)-O-(2-N-acetyl-2-
deoxy-β-D-glucopyranosyl)-(1f4)-O-(β-D-glucopyranosyluronic
acid)-(1f3)-O-(2-N-acetyl-2-deoxy-β-D-glucopyranosyl)-(1f4)-
O-(β-D-glucopyranosyluronic acid)-(1f3)-2-N-acetyl-2-deoxy-
β-D-glucopyranoside (9). Compound 10 was converted to 29
following the general procedure for TBS removal as described
9 (6.6 mg, 3.5 μmol, 35% from 29 for three steps): [R]20 -27
D
(c 0.05, H2O); 1H NMR (500 MHz, D2O) δ 1.90-1.92 (m, 15 H,
NHCOCH3 ꢀ 5), 3.20-3.26 (m, 5 H), 3.35-3.43 (m, 15 H),
3.45-3.48 (m, 5 H), 3.56-3.66 (m, 20 H), 3.70-3.82 (m, 10 H),
4.34 (d, 2 H, J = 8.5 Hz, anomeric H ꢀ 2), 4.36 (d, 4 H, J = 7.0 Hz,
anomeric H ꢀ 4), 4.44 (d, 2 H, J = 8.5 Hz, anomeric H ꢀ 2),
4.45 (d, 2 H, J= 7.5 Hz, anomeric H ꢀ 2); HRMS [M - 3H]3- m/z
calcd for C71H106N5O56 641.5200, found 641.5198.
Acknowledgment. We are grateful for the National Insti-
tutes of Health (R01-GM-72667) and a CAREER award
from the National Science Foundation (CHE0852308) for
financial support of this work.
Supporting Information Available: General experimental
1
procedures and selected H, 13C, and 2D NMR spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
J. Org. Chem. Vol. 74, No. 20, 2009 7617