Toward the modular synthesis of glycosaminoglycans: Synthesis of hyaluronic acid disaccharide building blocks using a periodic acid oxidation

Article abstract of DOI:10.1016/j.tet.2004.06.037

The synthesis of two differentially protected GluNAc-β(1→4)-GluA and GluA-β(1→3)-GluNAc disaccharide modules for the solid-phase assembly of hyaluronic acid are described. A periodic acid/chromium trioxide oxidation was the key transformation to facilitate access to the glucuronic acid moiety from glucose and should find wide application in the oxidation of primary alcohols.

Full text of DOI:10.1016/j.tet.2004.06.037

Tetrahedron 60 (2004) 7755–7766
Toward the modular synthesis of glycosaminoglycans:
synthesis of hyaluronic acid disaccharide building
blocks using a periodic acid oxidation
†
Emma R. Palmacci and Peter H. Seeberger
‡
,
*
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
Received 8 April 2004; revised 27 May 2004; accepted 1 June 2004
Available online 7 July 2004
Dedicated to Professor Dieter Seebach
Abstract—The synthesis of two differentially protected GluNAc-b(1!4)-GluA and GluA-b(1!3)-GluNAc disaccharide modules for the
solid-phase assembly of hyaluronic acid are described. A periodic acid/chromium trioxide oxidation was the key transformation to facilitate
access to the glucuronic acid moiety from glucose and should find wide application in the oxidation of primary alcohols.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
fied hyaluronic acid is used as a postoperative anti-adhesive
and as a biodegradable material for applications such as
drug delivery and tissue engineering.13 Hyaluronic acid, as
is characteristic of glycosaminoglycan structures, is a large,
linear repeating polymer of alternating hexosamine and
uronic acid monosaccharides (Fig. 1).3 Notably, HA is the
only unsulfated glycosaminoglycan and is comprised of
glucuronic acid b-(1!3) linked to N-acetyl glucosamine.
Glycosaminoglycans, the most acidic biopolymers, are part
of the extracellular matrixproteoglycan and cell surface, and
are found intracellularly in granules.1 Ubiquitous in the
human body, glycosaminoglycans play a key role in
regulating the biological activity of several proteins in the
coagulation cascade in addition to many other processes of
biomedical importance including growth factor interactions,
virus entry, and angiogenesis.2,3
Access to defined oligosaccharides is crucial to under-
standing the role of hyaluronic acid in biological systems.
Current methods for the isolation of hyaluronic acid rely on
the degradation of polymers from animal sources or from
enzymatic fermentation, resulting in a broad molecular
weight distribution and sample contamination with other
glycosaminoglycans.4,14 Chemoenzymatic approaches have
been applied to the acquisition of hyaluronic acid and
chondroitin sulfate with promising results.15 Still, the
procurement of useful quantities of defined glycosamino-
glycan structures relies mostly on synthetic methods.16–19
The glycosaminoglycan structure-activity relationship is still
poorly understood due to the complexity and heterogeneity of
these polymers. It has become increasingly evident that
defined lengths and sequences of glycosaminoglycans are
responsible for binding to a particular protein and modulating
its biological activity.4–7 Amoredetailedunderstandingofthe
structure-function relationships of glycosaminoglycans pro-
vides an opportunity for the discovery of novel therapeutic
interventions for a variety of disease states.
Heparin is the glycosaminoglycan studied in most detail, but
hyaluronic acid (HA) has seen increased interest due to its
involvement in wound healing,8 tumor metastasis,9 neuro-
degenerative diseases10 and arthritis.11,12 Clinically, modi-
2. Synthetic strategy
We are currently pursuing an overall program aimed at the
automated solid support synthesis of all naturally occurring
Keywords: Hyaluronic acid; Oxidation; Glycosaminoglycans; Modular
synthesis; Periodic acid.
*
Corresponding author. Tel.: þ41-1-633-2103; fax: þ41-1-633-1235;
e-mail address: seeberger@org.chem.ethz.ch
Thios Pharmaceuticals, Emeryville, CA.
†
‡
Laboratory for Organic Chemistry, ETH-Zurich/HCI F 315, Wolfgang-
Pauli-Strasse 10, CH-8093 Zu¨rich, Switzerland.
Figure 1. Hyaluronic acid.
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.06.037

7756
E. R. Palmacci, P. H. Seeberger / Tetrahedron 60 (2004) 7755–7766
Scheme 1. Different synthetic strategies for the assembly of hyaluronic acid oligosaccharides. (a) Monosaccharide approach; (b) disaccharide approach using
uronic acid reducing end; (c) disaccharide approach using glucosamine reducing end.
classes of glycoconjugates.20 Particular interest has been
focused on the modular assembly of glycosaminoglycans
including heparin.21 The automated synthesis of HA may be
envisioned using either monosaccharides (Scheme 1(a)) or
disaccharides (Scheme 1(b)or(c)). Twodifferentdisaccharide
modules can be used that contain either a terminal uronic acid
(Scheme 1(b)) or a terminal glucosamine (Scheme 1(c)).
oligosaccharides will require the preparation of differen-
tially protected glucosamine building blocks. In designing a
monomer that would be not only useful for the synthesis of
HA but that might also serve in the synthesis of chondroitin
sulfate oligomers, a benzoate group was installed on the
C4 hydroxyl group that could be removed at the oligo-
saccharide stage if desired. Synthesis of such a differentially
protected monomer utilized known tetraacetyl N-trichloro-
acetamide glucosamine 122 as starting material. The
anomeric acetate of glucosamine 1 was exchanged for an
anomeric silyl ether. Removal of the remaining acetates and
installation of a 4,6-di-O-benzylidene afforded monosac-
charide 2 (Scheme 2). Protection of the C3-hydroxyl as a
levulinic ester and opening of the 4,6-benzylidene under
reductive conditions (TES, TFA) afforded the C4-OH
glucosamine 3. Benzoylation of the liberated hydroxyl
group afforded fully protected 4. Conversion of 4 to
glycosyl trichloroacetimidate 5 by cleavage of the anomeric
silyl ether with TBAF and subsequent treatment of the lactol
with trichloroacetonitrile and catalytic amounts of DBU was
readily achieved.
Efficient glycosyl building blocks to be used in an
automated assembly scheme, whether using the monosac-
charide or disaccharide approach, must meet several
criteria. The activation and deprotection conditions must
be compatible with the linker and the solid support. Previous
work with the automated synthesis of oligosaccharides
demonstrated that trichloroacetimidates and glycosyl phos-
phates work well with the automated method, along with
acetate, levulinate and silyl ethers temporary protecting
groups.20 The incompatibility of the glucuronic acid moiety
in the glycosaminoglycan structures precludes the use of
acetyl esters and silyl ethers, therefore the synthesis would
be based on the levulinate temporary protecting group.22
The amine protecting group chosen was the N-trichloro-
acetamide group, based on its successful use in previous
synthesis requiring the glycosylation of the C3-hydroxyl of
glucosamine and its direct transformation to the necessary
N-acetamide.23,24
4. Synthesis of a differentially protected glucuronic acid
building block
In addition to the glucosamine monomer, straightforward
access to a glucuronic acid monosaccharide is essential
to synthesize hyaluronic acid. In previous syntheses,
lengthy procedures were used to access differentially
protected glucuronic acid modules.16–19 For use in a
disaccharide approach, a glucuronic acid acceptor would
be equipped with a temporary protecting group at C1, an
ester at C2 to confer stereochemistry in the glycosylation
reaction and a C3-O-benzyl group to ensure high
coupling yields.
Following these principal strategic considerations the
synthesis of the required building blocks to validate the
feasibility of each approach was undertaken.
3. Synthesis of a differentially protected glucosamine
building block
The three different strategies for the assembly of defined HA
Scheme 2. Synthesis of glucosamine building block 5.

E. R. Palmacci, P. H. Seeberger / Tetrahedron 60 (2004) 7755–7766
7757
Scheme 3. Synthesis of diols for selective oxidation.
Table 1. Attempted oxidation of 7, 8 and 11
The synthesis of the glucuronic acid module was attempted
initially through a selective glucose oxidation. TEMPO was
reported to selectively oxidize primary hydroxyls, giving
the corresponding uronic acid.23,24 The direct conversion of
a diol to the glucuronic acid hydroxyl acceptor would
eliminate much of the protecting group manipulations that
plagued previous syntheses of glucuronic acid.
Starting material
Oxidation conditions
Yield (%)
7
TEMPO/NaOCl
Pt/C or Pd/C, O2
20
0
8
TEMPO/NaOCl
Pt/C or Pd/C, O2
20
0
11
TEMPO/NaOCl
PDC/Ac2O
PDC/MS
0
0
0
Two differentially protected glucoses were studied for the
oxidation to the corresponding C5-carboxylic acids
(Scheme 3). Diols 7 and 8 were synthesized using known
procedures and submitted to TEMPO oxidation conditions.
The oxidations did not yield more than 20% of the desired
compounds. Platinum25,26 and palladium27 on carbon in the
presence of oxygen have been reported to selectively
oxidize primary alcohols to acids. Refluxing diols 7 and 8
in dioxane/water in the presence of the metal catalyst and O2
resulted in decomposed starting material.
as an acetyl ester and removal of the PMB ether resulted in
differentially protected glucose 12.
Several methods for the oxidation of 12 were evaluated in
the synthesis of the desired glucuronic acid (Table 2).
Oxidation with PDC required six equivalents of the
chromium oxidant and the difficult purification yielded
only small amounts of the desired product. Use of resin-
bound PDC was failed to remedy this problem as the
support-bound reagent was too unreactive to facilitate the
transformation.30,31 TEMPO mediated oxidation with
sodium hypochlorite was incompatible with allyl ether
protected carbohydrates.32 Attempts to oxidize the C6-
hydroxyl group to the corresponding aldehyde via Swern
oxidation33 using either oxalyl chloride afforded the a,b-
unsaturated aldehyde 14 as the major product due to
elimination of the C4-O-acetate. The oxidation reagents
Non-selective oxidation conditions with differentially
protected glucose derivatives were examined next. Initial
results with the silyl and methoxyphenyl anomeric protected
monosaccharides revealed that these modes of protection
were not stable under oxidizing conditions. The synthesis of
allyl glycosides 10–13 was readily achieved by protecting
group manipulations starting from 4,6-p-methoxybenzyli-
dene protected glucose 9. Installation of the C2-pivaloyl,
followed by regioselective opening of the 4,6-benzylidene
with NaCNBH3 and TMS-Cl afforded C4-p-methoxybenzyl
11 (Scheme 4). Treatment of 11 with pyridinium dichromate
(PDC)28,29 or TEMPO in attempts to effect oxidation to the
corresponding glucuronic acid resulted in decomposition of
the PMB ether (Table 1). Further protecting group
adjustments were needed to acquire a suitably protected
glucose starting material. Treatment of 10 with NaCNBH3
and trifluoroacetic acid afforded the corresponding C6-p-
methoxylbenzyl ether. Masking of the C4-hydroxyl group
TPAP,34 RuCl2(PPh)3 and NaNO2/Ac2O36 failed to react
with the starting materials.
35
5. Successful oxidation and synthesis of the
glucosamine–b(1!4)-glucuronic acid disaccharide
A chromium trioxide oxidation was reported to have
Scheme 4. Synthesis of allyl protected glucoses.

7758
E. R. Palmacci, P. H. Seeberger / Tetrahedron 60 (2004) 7755–7766
Table 2. Oxidation of 12
With the glucosamine portion of a putative disaccharide
module in hand, we undertook the preparation of the glucose
that at the disaccharide stage would become the glucuronic
acid moiety by periodic acid oxidation. Thus, differentially
protected glucose thioglycoside 22 was synthesized from
known 2143 (Scheme 7) by selective opening of the 4,6-p-
methoxybenzylidene ring and protection of the resulting
hydroxyl. The anomeric thioether served to protect the
anomeric position during the installation of the necessary
protecting group and was utilized subsequently as glycosy-
lating agent. Activation of 22 with MeOTf in the presence of
20 initiated the union to afford the corresponding dis-
accharide (Scheme 7). Treatment of the crude reaction
mixture with ceric ammonium nitrate resulted in the
removal of the PMB ether and provided disaccharide 23
in 50% yield. Oxidation of 23 with H5IO6/CrO3 in
acetonitrile and subsequent methylation afforded 24
(39%), still accompanied by unreacted disaccharide 23.
An increase in reaction time and equivalents of the
oxidizing solution did not improve the conversion to the
carboxylic acid. Nevertheless, access to the reducing end
disaccharide module 24 that in addition can be readily
converted into a repeating disaccharide module was
achieved.
Starting material
Oxidation conditions
Yield (%)
12
PDC/Ac2O
TPAP, O2, ms
TEMPO/NaOCl
20
0
0
0
PDC
Oxalyl-CI, DMSO, Et3N
H5IO6, CrO3
0a
70b
a
Oxidation yielded 14 but 0% of desired compound.
Yield of 15 after methylation.
b
excellent functional group tolerance under conditions that
would be suitable for a carbohydrate scaffold.37 – 39
Oxidation of 12 with a solution of periodic acid and a
catalytic amount of chromium trioxide40 in wet acetonitrile
cleanly afforded the corresponding glucuronic acid (Table
2). Formation of the methyl ester was accomplished with
methyl iodide and NaHCO3 in DMF to furnish 15 in 70%
yield. Treatment of 15 with sodium methoxide in methanol
yielded the desired glucuronic acid allyl glycoside 16 and
the elimination product in approximately equal amounts. An
improved pathway to 16 commenced with C4-levulinate
protected 13. Oxidation with H5IO6/CrO3 and removal the
levulinate protecting group cleanly afforded acceptor 16.
Glycosylation of 16 with glucosamine 5 afforded HA
disaccharide 17 in 86% yield (Scheme 5). The disaccharide
module 17 can serve either as reducing end disaccharide or
as precursor for the preparation of a repeating disaccharide
glycosylating agent that would merely require deprotection
and installation of an anomeric leaving group.
7. Summary
Described is the synthesis of several differentially protected
glucosamine and glucuronic acid monosaccharides as well
as two HA disaccharide modules that constitute key
building blocks for the assembly of defined hyaluronic
acid oligomers. A mild periodic acid oxidation protocol to
operate on a glucose monosaccharide and a glucose–
b(1!3)-glucosamine disaccharide to install the glucuronic
acid moiety was developed. This transformation had been
difficult or impossible with a long list of other oxidation
procedures but was readily achieved applying a mild
periodic acid oxidation. Future work will evaluate both
disaccharides in the synthesis of hyaluronic acid oligomers
in solution and on solid support. The periodic acid oxidation
procedure should prove useful in the synthesis of other
carboxylic acid containing carbohydrates.
6. Synthesis of glucuronic acid–b(1!3)-glucosamine
disaccharide
In addition to disaccharide module 17 that may be used in
the synthesis of HA oligosaccharides as outlined in
Scheme 1, the reverse disaccharide module containing a
glucosamine reducing end also was accessible utilizing the
periodic acid oxidation. Allyl glycoside 1841 was employed
for the synthesis of the glucosamine monosaccharide since
this anomeric protecting group proved to be compatible with
the periodic acid C6 oxidation. Protection of the C3-
hydroxyl of 1842 with levulinic acid and opening of the 4,6-
benzylidene yielded 19 (Scheme 6). Acetylation and
treatment with hydrazine acetate afforded differentially
protected glucosamine 20 ready for further elaboration.
8. Experimental
8.1. General methods
All chemicals used were reagent grade and used as supplied
except where noted. Dichloromethane (CH2Cl2) and tetra-
hydrofuran (THF) and toluene were purchased from JT
Baker and purified by a Cycle-Tainer Solvent Delivery
System. Pyridine was refluxed over calcium hydride and
distilled prior to use. Trimethylsilyl trifluoromethanesulfo-
nate (TMSOTf) was purchased from Acros Chemicals.
Analytical thin-layer chromatography was performed on
E. Merck silica gel 60 F254 plates (0.25 mm). Compounds
were visualized by dipping the plates in a cerium sulfate-
ammonium molybdate solution followed by heating. Liquid
chromatography was performed using forced flow of the
1
indicated solvent on Sigma H-type silica (10–40 mm). H
NMR spectra were obtained on a Varian VXR-300
Scheme 5. Construction of the disaccharide building block.

E. R. Palmacci, P. H. Seeberger / Tetrahedron 60 (2004) 7755–7766
7759
Scheme 6. Synthesis of allyl protected glucosamine 20.
Scheme 7. Synthesis of thioglycoside 22 and disaccharide 24.
(300 MHz), Varian VXR-500 (500 MHz) or a Bruker 400
(400 MHz) and are reported in parts per million (d) relative
to CHCl3 (7.27 ppm). Coupling constants (J) are reported in
Hertz. 13C NMR spectra were obtained on a VXR-300
(75 MHz), a Bruker-400 (100 MHz) or a VXR-500
(125 MHz, CDCl3) and are reported in d relative to
CDCl3 (77.23 ppm) as an internal reference. 31P spectra
were obtained on a VXR-300 (120 MHz) and are reported in
d relative to H3PO4 (0.0 ppm) as an external reference.
trichloroacetamido-b-D-glucopyranoside(2.70 g,4.78 mmol)
was dissolved in MeOH (30 mL) and cooled to 0 8C.
Sodium methoxide (328 mL, 1.43 mmol, 25 wt% solution in
MeOH) was added and the resulting solution was stirred for
2 h at 0 8C. Dowex acidic resin was added to adjust the pH
to 7, the resin was filtered off and the solvents were removed
in vacuo. The residue was dissolved in CH3CN (40 mL) and
benzaldehyde dimethylacetal (0.96 mL, 6.20 mmol) and
p-toluenesulfonic acid (25 mg) were added. The reaction
mixture was stirred for 1 h, diluted with EtOAc (100 mL)
and washed with water (1£100 mL) and sat. NaHCO3
(1£100 mL). The organic layer was dried over Na2SO4,
filtered and concentrated to afford tert-butyldimethylsilyl
4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-b-D-glu-
copyranoside 2; IR (thin film, NaCl) 3332, 2858, 1696,
8.1.1. tert-Butyldimethylsilyl 3,4,6-tri-O-acetyl-2-deoxy-
2-trichloroacetamido-b-D-glucopyranoside.
1,3,4,6-
Tetra-O-acetyl-2-deoxy-2-trichloroacetamido-D-glucopyra-
noside (1.18 g, 2.40 mmol) was dissolved in THF (20 mL).
Benzyl amine (0.80 mL, 7.20 mmol) was added and the
solution was stirred for 6 h. The mixture was diluted with
EtOAc (100 mL) and washed with 5% HCl (100 mL). The
aqueous layer was extracted with EtOAc (3£100 mL) and
the combined organic layers were dried over Na2SO4,
filtered, and the solvents were removed in vacuo. The
residue was dissolved in DMF (15 mL) and TBS chloride
(0.470 g, 3.12 mmol) and imidazole (0.293 g, 4.80 mmol)
were added. The solution was stirred for 12 h, diluted with
Et2O (100 mL) and washed with sat. NaHCO3 (100 mL).
The organic layer was dried over Na2SO4, filtered, and the
solvents were removed in vacuo. Chromatography (3:1
hexanes–EtOAc) afforded tert-butyldimethylsilyl 3,4,6-tri-
O-acetyl-2-deoxy-2-trichloroacetamido-b-D-glucopyrano-
side as a colorless oil (0.950 g, 70%). [a]2D4¼24.28 (c 2.62,
CH2Cl2); IR (thin film, NaCl) 2931, 2858, 1751, 1717,
1
840 cm21; H NMR (500 MHz, CDCl3) d 7.50–7.48 (m,
2H), 7.40–7.34 (m, 2H), 6.98 (d, J¼7.0 Hz, 1H), 5.54 (s,
1H), 5.11 (d, J¼7.9 Hz, 1H), 4.32–4.27 (m, 2H), 3.79 (app
t, J¼10.1 Hz, 1H), 3.59–3.48 (m, 3H), 0.89 (s, 9H), 0.13 (s,
3H), 0.12 (s, 3H); 13C NMR (125 MHz, CDCl3) d 162.9,
137.6, 130.9, 130.1, 129.2, 129.1, 127.0, 102.6, 95.9, 82.3,
70.3, 69.3, 66.9, 62.3, 26.3, 18.5, 23.4, 24.5; ESI MS m/z
(M)þ: calcd 526.2 obsd 526.9. 2 was dissolved in CH2Cl2
(30 mL) and levulinic anhydride (2.05 g, 9.56 mmol) and
DMAP (0.700 g, 5.74 mmol) were added. The reaction
mixture was stirred for 30 min and diluted with CH2Cl2
(50 mL) and the organic layer was washed with sat.
NaHCO3 (1£100 mL). The aqueous layer was extracted
with CH2Cl2 (2£100 mL) and the combined organic layers
were dried over Na2SO4, filtered and concentrated.
Chromatography (5:1 hexanes–EtOAc) afforded tert-butyl-
dimethylsilyl 4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-
2-trichloroacetamido-b-D-glucopyranoside as a colorless
syrup (2.30 g, 76%). [a]2D4¼223.98 (c 2.14, CH2Cl2); IR
1230 cm21
;
1H NMR (500 MHz, CDCl3) d 6.84 (d,
J¼8.8 Hz, 1H), 5.31 (dd, J¼9.2, 9.5 Hz, 1H), 5.09 (app t,
J¼9.5 Hz, 1H), 4.87 (d, J¼7.9 Hz, 1H), 4.22 (dd, J¼2.4,
12.2 Hz, 1H), 3.98–3.93 (m, 1H), 3.74 (ddd, J¼2.4, 6.1,
9.8 Hz, 1H), 2.08 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H); 13C
NMR (125 MHz, CDCl3) d 171.3, 170.8, 169.5, 162.0, 96.0,
92.5, 72.2, 71.8, 68.9, 62.6, 58.1, 25.7, 20.9, 20.8, 20.8, 18.0,-
4.0, 25.1; ESI MS m/z (MþNH4)þ: calcd 581.0 obsd 581.4.
1
(thin film, NaCl) 2930, 2858, 1719, 1103 cm21; H NMR
(500 MHz, CDCl3) d 7.49–7.47 (m, 2H), 7.39–7.35 (m,
2H), 7.15 (d, J¼9.1 Hz, 1H), 5.51 (s, 1H), 5.44 (app t,
J¼9.8 Hz, 1H), 4.86 (d, J¼8.1 Hz, 1H), 4.11 (dd, J¼5.2,
10.7 Hz, 1H), 4.01–3.96 (m, 1H), 3.76–3.70 (m, 2H), 3.47
(app td, J¼5.2, 9.8 Hz, 1H), 2.71 (app td, J¼3.4, 7.3 Hz,
2H), 2.60 (t, J¼6.4 Hz, 2H), 2.14 (s, 3H), 0.87 (s, 9H), 0.08
(s, 3H), 0.07 (s, 3H); 13C NMR (125 MHz, CDCl3) d 206.1,
8.1.2. tert-Butyldimethylsilyl 4,6-O-benzylidene-2-deoxy-
3-O-levulinoyl-2-trichloroacetamido-b-D-glucopyrano-
side 2. tert-Butyldimethylsilyl 3,4,6-tri-O-acetyl-2-deoxy-2-

7760
E. R. Palmacci, P. H. Seeberger / Tetrahedron 60 (2004) 7755–7766
173.2, 162.2, 137.2, 129.3, 128.4, 126.3, 101.5, 96.5, 92.7,
78.9, 77.5, 77.0, 71.5, 68.7, 66.6, 58.8, 38.2, 30.0, 28.3,
25.8, 18.0, 23.9, 24.9; ESI MS m/z (MþNH4)þ: calcd
641.1 obsd 641.5.
0.328 mmol) were added simultaneously. After 1 h, EtOAc
(20 mL) was added and the organic layer was washed with
water (20 mL). The aqueous layer was extracted with
EtOAc (3£20 mL) and the combined organic layers were
washed with sat. NaHCO3 (1£50 mL), dried over Na2SO4,
filtered and concentrated. The residue was dissolved in
CH2Cl2 (2.0 mL) and the solution was cooled to 0 8C.
Trichloroacetonitrile (1.0 mL) was added followed by the
addition of DBU (25 mL). The reaction mixture was stirred
for 1 h, filtered through a silica plug (3:1 hexanes–EtOAc)
and the solvents were removed in vacuo to afford 5 as a
yellow foam (0.254 g, 98%). [a]2D4¼þ55.78 (c 4.35,
CH2Cl2); IR (thin film, NaCl) 3064, 2960, 1728,
8.1.3. tert-Butyldimethylsilyl 6-O-benzyl-2-deoxy-3-O-
levulinoyl-2-trichloroacetamido-b-D-glucopyranoside
3. tert-Butyldimethylsilyl 4,6-O-benzylidene-2-deoxy-
3-O-levulinoyl-2-trichloroacetamido-b-D-glucopyranoside
(0.710 g, 1.14 mmol) was co-evaporated with toluene
(3£10 mL), dissolved in CH2Cl2 (15 mL) and cooled to
0 8C. Triethylsilane (0.910 mL, 5.70 mmol) was added,
followed by the slow addition of trifluoroacetic acid
(0.440 mL, 5.70 mmol). After 3 h, the reaction mixture
was diluted with EtOAc (50 mL). The organic layer was
washed with water (50 mL) and sat. NaHCO3 (50 mL),
dried over Na2SO4, filtered and concentrated. Chromatog-
raphy (3:2!1:2 hexanes–EtOAc) afforded 3 (0.526 g,
75%). [a]2D4¼228.28 (c 1.57, CH2Cl2); IR (thin film,
1
1266 cm21; H NMR (500 MHz, CDCl3) d 8.84 (s, 1H),
7.97 (d, J¼7.6 Hz, 2H), 7.59 (t, J¼7.3 Hz, 1H), 7.46 (t,
J¼7.6 Hz, 2H), 7.23–7.16 (m, 4H), 7.10 (d, J¼8.2 Hz, 1H),
6.58 (d, J¼3.4 Hz, 1H), 5.67 (app t, J¼9.8 Hz, 1H), 5.60
(app t, J¼9.8 Hz, 1H), 4.54–4.44 (m, 4H), 4.26 (dt, J¼3.7,
9.8 Hz, 1H), 3.66 (dd, J¼2.7, 11.3 Hz, 1H), 3.60 (dd, J¼4.3,
11.3 Hz, 1H), 2.62–2.59 (m, 2H), 2.55–2.46 (m, 2H), 2.01
(s, 3H); 13C NMR (125 MHz, CDCl3) d 205.6, 173.6, 165.1,
162.2, 160.2, 137.5, 133.8, 130.1, 129.1, 129.0, 128.7,
128.4, 128.0, 128.0, 127.8, 94.0, 91.9, 90.8, 73.8, 72.2, 70.7,
68.1, 68.0, 54.3, 37.8, 29.6, 28.2; ESI MS m/z (MþNH4)þ:
calcd 600.4 obsd 600.8.
NaCl) 2956, 2858, 1742, 1717, 1530 cm21 1H NMR
;
(400 MHz, CDCl3) d 7.36–7.26 (m, 4H), 7.05 (br s, 1H),
5.26–5.20 (m, 1H), 4.83 (d, J¼7.9 Hz, 1H), 4.61 (d,
J¼12.1 Hz, 1H), 4.57 (d, J¼12.1 Hz, 1H), 3.98–3.89 (m,
1H), 3.80–3.73 (m, 3H), 3.66–3.61 (m, 1H), 3.33 (br s, 1H),
2.78 (app t, J¼6.4 Hz, 1H), 2.63–2.44 (m, 2H), 2.17 (s, 3H),
0.87 (s, 9H), 0.13 (s, 3H), 0.09 (s, 3H); 13C NMR (100 MHz,
CDCl3) d 207.9, 173.6, 162.1, 138.1, 128.6, 127.9, 127.8,
96.1, 92.7, 75.6, 74.5, 70.4, 70.0, 57.7, 38.5, 30.0, 28.5,
25.8, 18.0, 23.9, 25.0; ESI MS m/z (MþNH4)þ: calcd
643.1 obsd 643.6.
8.1.6. p-Methoxyphenyl 3-O-benzyl-4,6-di-O-benzyli-
dene-b-D-glucopyranoside 6. p-Methoxyphenyl 2,4,6-tri-O-
acetyl-3-O-benzyl-b-D-glucopyranoside (1.16 g, 2.31 mmol)
was dissolved in MeOH (25 mL) and NaOMe (25% wt in
MeOH, 210 mL, 0.924 mmol) was added. After 3 h, the
reaction mixture was neutralized with the addition of
Dowex acid resin, filtered and the solvents were removed
in vacuo. The residue was dissolved in CH3CN (25 mL) and
DMF (2.0 mL). Benzylidene dimethylacetal (430 mL,
2.77 mmol) and p-toluenesulfonic acid (50 mg) was
added. After 15 h, the solid precipitate was collected on a
fritted funnel to afford 6 (810 mg, 75%); IR (thin film,
8.1.4. tert-Butyldimethylsilyl 4-O-benzoyl-6-O-benzyl-2-
deoxy-3-O-levulinoyl-2-trichloroacetamido-b-D-gluco-
pyranoside 4. tert-Butyldimethylsilyl 6-O-benzyl-2-deoxy-
3-O-levulinoyl-2-trichloroacetamido-b-D-glucopyranoside 3
(1.02 g, 1.63 mmol) was dissolved in CH2Cl2 (15 mL) and
benzoyl chloride (0.23 mL, 1.95 mmol) and DMAP
(0.240 g, 1.95 mmol) were added. After 1 h, hexanes–
EtOAc (3:1, 50 mL) was added and the white precipitate
was removed by filtration through a silica plug. The solvents
were removed in vacuo and chromatography (3:1!3:2
hexanes–EtOAc) afforded 4 as a colorless oil (1.09 g, 91%);
IR (thin film, NaCl) 2956, 2930, 1724, 1271 cm21; 1H NMR
(500 MHz, CDCl3) d 7.95 (d, J¼8.4 Hz, 2H), 7.60–7.53 (m,
2H), 7.44 (t, J¼7.9 Hz, 1H), 7.25–7.20 (m, 4H), 6.81 (d,
J¼8.9 Hz, 1H), 5.54 (dd, J¼9.2, 10.7 Hz, 1H), 5.35 (app t,
J¼9.5 Hz, 1H), 5.07 (d, J¼7.9 Hz, 1H), 4.52 (d, J¼12.2 Hz,
1H), 4.49 (d, J¼12.2 Hz, 1H), 3.91–3.84 (m, 2H), 3.63 (d,
J¼4.3 Hz, 2H), 2.58–2.56 (m, 2H), 2.47–2.39 (m, 2H),
2.02 (s, 3H), 0.90 (s, 9H), 0.17 (s, 3H), 0.13 (s, 3H); 13C
NMR (100 MHz, CDCl3) d 206.1, 172.7, 170.2, 165.5,
162.0, 137.8, 133.8, 133.7, 130.3, 130.0, 129.3, 128.7,
128.4, 127.8, 95.7, 92.5, 73.8, 73.7, 71.9, 69.9, 69.5, 58.7,
38.0, 29.7, 28.2, 25.8, 18.0, 23.9, 25.1; ESI MS m/z
(MþNH4)þ: calcd 747.2 obsd 747.4.
1
NaCl) 3479, 2869, 2361, 1734 cm21; H NMR (300 MHz,
CDCl3) d 7.55–7.27 (m, 5H), 7.03–6.82 (m, 5H), 5.61 (s,
1H), 5.01 (d, J¼12.0 Hz, 1H), 4.91 (d, J¼7.2 Hz, 1H), 4.83
(d, J¼11.7 Hz, 1H), 4.38 (dd, J¼4.8, 10.2 Hz, 1H), 3.88–
3.81 (m, 5H), 3.72–3.50 (m, 2H), 2.37 (br s, 1H); 13C NMR
(125 MHz, CDCl3) d 129.3, 128.9, 128.7, 128.5, 128.3,
128.3, 128.3, 128.1, 126.2, 118.9, 118.3, 114.9, 114.8,
102.7, 101.5, 83.7, 81.4, 80.4, 75.6, 75.1, 74.9, 74.6, 74.2,
70.3, 68.9, 66.8, 62.8, 55.9; ESI MS m/z (M)þ: calcd 464.1
obsd 463.3.
8.1.7. p-Methoxyphenyl 3-O-benzyl-2-O-pivaloyl-b-D-
glucopyranoside 7. p-Methoxyphenyl 3-O-benzyl-4,6-di-O-
benzylidene-b-D-glucopyranoside 6 (810 mg, 1.75 mmol)
was dissolved in CH2Cl2 (20 mL) and pivaloyl chloride
(257 mL, 2.10 mmol) and DMAP (427 mg, 3.50 mmol)
were added. After 30 min, the reaction mixture was diluted
with CH2Cl2 (20 mL) and the organic layer was washed
with sat. NaHCO3 (50 mL). The aqueous layer was
extracted with CH2Cl2 (3£50 mL) and the combined
organic layers were washed with 5% HCl (150 mL), dried
over Na2SO4, filtered and the solvents removed in vacuo. The
crude mixture was dissolved in hexanes–EtOAc (3:1, 75 mL)
and the solution was cooled to 225 8C. The white precipitate
was collected and dried to afford p-methoxyphenyl 3-O-
8.1.5. 4-O-Benzoyl-6-O-benzyl-2-deoxy-3-O-levulinoyl-
2-trichloroacetamido-a-D-glucopyranosyl trichloroace-
timidate 5. tert-Butyldimethylsilyl 4-O-benzoyl-6-O-ben-
zyl-2-deoxy-3-O-levulinoyl-2-trichloroacetamido-b-D-glu-
copyranoside 4 (0.200 g, 0.274 mmol) was dissolved in
THF (3.0 mL) and the solution was cooled to 0 8C. TBAF
(0.33 mL, 1 M solution in THF) and AcOH (18.7 mL,

E. R. Palmacci, P. H. Seeberger / Tetrahedron 60 (2004) 7755–7766
7761
benzyl-4,6-di-O-benzylidene-2-O-pivaloyl-b-D-glucopyra-
noside (798 mg, 85%); 1H NMR (300 MHz, CDCl3) d
7.49–7.24 (m, 5H), 6.95–6.78 (m, 4H), 5.59 (s, 1H), 5.30
(app t, J¼8.3 Hz, 1H), 4.96 (d, J¼8.0 Hz, 1H), 4.88 (d,
J¼11.6 Hz, 1H), 4.68 (d, J¼11.8 Hz, 1H), 4.39 (dd,
J¼4.9, 10.7 Hz, 1H), 3.91–3.80 (m, 3H), 3.77 (s, 3H),
3.60–3.54 (m, 2H), 1.20 (s, 9H). p-Methoxyphenyl 3-O-
benzyl-4,6-di-O-benzylidene-2-O-pivaloyl-b-D-glucopyra-
noside (798 mg, 1.46 mmol) was dissolved in CH2Cl2
(10 mL) and the solution was cooled to 0 8C. Trifluoro-
acetic acid (400 mL) and water (600 mL) were added and
the solution was allowed to warm and stir over 4 h.
CH2Cl2 (30 mL) was added and the organic layer was
washed with sat. NaHCO3 (50 mL). The aqueous layer
was extracted with CH2Cl2 (3£40 mL), the organic layers
were dried over Na2SO4, filtered and the solvents were
removed in vacuo. Column chromatography (3:2 hex-
anes–EtOAc) afforded 7 as a glassy solid (618 mg, 92%);
side (136 mg, 0.245 mmol) was dissolved in CH2Cl2
(2.0 mL) and water (40 mL). Trifluoroacetic acid
(57.0 mL, 0.735 mmol) was added dropwise and the
resulting solution was stirred for 4 h. The reaction mixture
was diluted with CH2Cl2 (10 mL) and the organic layer was
washed with sat. NaHCO3 (10 mL). The aqueous layers
were extracted with CH2Cl2 (3£10 mL), the combined
organic layers were dried over MgSO4, filtered and the
solvents were removed in vacuo. Column chromatography
1
(5:1!3:2 hexanes–EtOAc) afforded 8 (60.5 mg, 52%); H
NMR (500 MHz, CDCl3) d 7.32–7.27 (m, 5H), 4.99 (d,
J¼7.5, 7.6 Hz, 1H), 4.78 (d, J¼11.6 Hz, 1H), 4.75 (d,
J¼7.6 Hz, 1H), 4.58 (d, J¼11.6 Hz, 1H), 3.91–3.86 (m,
1H), 3.78–3.74 (m, 1H), 3.69 (app dt, J¼3.1, 9.3 Hz, 1H),
3.55 (app t, J¼9.2 Hz, 1H), 3.42–3.39 (m, 1H), 2.20 (d,
J¼2.8 Hz, 1H), 1.93 (t, J¼6.6 Hz, 1H), 1.23 (s, 9H), 0.89 (s,
9H), 0.12 (s, 3H), 0.10 (s, 3H); 13C NMR (125 MHz,
CDCl3) d 176.9, 138.2, 128.9, 128.3, 127.8, 96.4,83.2, 75.3,
74.8, 74.4, 70.7, 63.0, 39.0, 27.5, 25.8, 18.0, 23.7, 24.6;
ESI MS m/z (MþNH4)þ: calcd 486.2 obsd 486.6.
IR (thin film, NaCl) 3402, 2967, 2931, 1738, 1508 cm21
;
1H NMR (500 MHz, CDCl3) d 7.37–7.27 (m, 5H), 6.92–
6.90 (m, 2H), 6.82–6.80 (m, 2H), 5.27 (dd, J¼7.9,
9.5 Hz, 1H), 4.93 (d, J¼7.9 Hz, 1H), 4.79 (d, J¼11.3 Hz,
1H), 4.65 (d, J¼11.6 Hz, 1H), 3.93 (dd, J¼3.4, 12.2 Hz,
1H), 3.81 (dd, J¼5.2, 12.2 Hz, 1H), 3.77 (s, 3H), 3.64
(app t, J¼9.2 Hz, 1H), 3.55–3.49 (m, 1H), 1.21 (s, 9H);
13C NMR (125 MHz, CDCl3) d 177.1, 155.5, 151.3,
138.1, 128.9, 128.3, 127.9, 118.0, 114.8, 100.6, 82.9,
75.6, 74.8, 72.8, 70.3, 62.6, 55.8, 39.1, 27.4.
8.1.10. Allyl 3-O-benzyl-4,6-di-O-p-methoxybenzylidene-
2-O-pivaloyl-b-D-glucopyranoside 10. Allyl 3-O-benzyl-
4,6-di-O-p-methoxybenzylidene-b-D-glucopyranoside
9
(1.00 g, 2.34 mmol) was dissolved in CH2Cl2 (20 mL) and
pivaloyl chloride (340 mL, 2.80 mmol) and DMAP
(630 mg, 5.85 mmol) were added. After 1 h, water (1 mL)
was added and the solution was diluted with CH2Cl2
(20 mL) and the organic layer was washed with sat.
NaHCO3 (50 mL), dried over MgSO4, filtered and the
solvents were removed in vacuo. Column chromatography
(10:1!3:1 hexanes–EtOAc) afforded 10 (1.10 g, 92%); IR
8.1.8. tert-Butyldimethylsilyl 3-O-benzyl-4,6-di-O-benzy-
lidene-2-O-pivaloyl-b-D-glucopyranoside.
p-Methoxy-
phenyl 3-O-benzyl-4,6-di-O-benzylidene-2-O-pivaloyl-b-
D-glucopyranoside (167 mg, 0.3 mmol) was dissolved in
CH3CN (4.0 mL) and water (1.0 mL). Ceric ammonium
nitrate (822 mg, 1.50 mmol) was added and the reaction
mixture was stirred for 30 min. The mixture was diluted
with EtOAc (10 mL) and the organic layer was washed with
sat. NaHCO3 (10 mL). The aqueous layers were extracted
with EtOAc (3£10 mL) and the combined organics were
dried over MgSO4, filtered and the solvents removed in
vacuo. The residue was dissolved in DMF (3.0 mL) was
tert-butyldimethylsilyl chloride (60 mg, 0.388 mmol) and
imidazole (40 mg, 0.60 mmol) were added. After 15 h, the
reaction mixture was diluted with EtOAc (10 mL) and
the organic layer was washed with sat. NaHCO3 (10 mL).
The aqueous layers were extracted with EtOAc (3£10 mL)
and the combined organics were dried over MgSO4, filtered
and the solvents removed in vacuo. Column chromato-
graphy (5:1 hexanes–EtOAc) afforded tert-butyldimethyl-
silyl 3-O-benzyl-4,6-di-O-benzylidene-2-O-pivaloyl-b-D-
glucopyranoside (137 mg, 82%); 1H NMR (400 MHz,
CDCl3) d 7.50–7.27 (m, 5H), 5.58 (s, 1H), 5.04 (dd,
J¼7.6, 8.8 Hz, 1H), 4.90 (d, J¼11.6 Hz, 1H), 4.81 (d,
J¼7.5 Hz, 1H), 4.66 (d, J¼11.6 Hz, 1H), 4.33 (dd, J¼5.0,
10.5 Hz, 1H), 3.86–3.73 (m, 3H), 3.53–3.46 (m, 1H), 1.19
(s, 9H), 0.94 (s, 9H), 0.12 (s, 3H), 0.10 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 176.8, 138.4, 137.4, 129.2, 128.5,
128.4, 127.6, 126.2, 101.4, 96.8, 81.8, 79.3, 74.8, 74.3, 68.9,
66.5, 38.9, 27.5, 25.9, 25.8, 18.0, 23.7, 23.8, 24.8.
1
(thin film, NaCl) 2971, 1736, 1518, 1097 cm21; H NMR
(500 MHz, CDCl3) d 7.40 (d, J¼8.9 Hz, 2H), 7.30–7.26 (m,
5H), 6.91 (d, J¼8.5 Hz, 2H), 5.86–5.76 (m, 1H), 5.54 (s,
1H), 5.29–5.17 (m, 1H), 5.10 (app t, J¼8.1 Hz, 1H), 4.86
(d, J¼11.8 Hz, 1H), 4.66 (d, J¼11.8 Hz, 1H), 5.54 (d,
J¼7.9 Hz, 1H), 4.36–4.31 (m, 1H), 4.05 (dd, J¼6.1,
12.7 Hz, 1H), 3.83–3.76 (m, 2H), 3.81 (s, 3H), 3.56–3.51
(m, 1H), 1.20 (s, 9H); 13C NMR (125 MHz, CDCl3) d 176.9,
160.2, 138.3, 133.5, 129.9, 128.4, 127.8, 127.7, 127.5,
117.9, 113.8, 101.4, 100.9, 81.6, 79.1, 74.4, 72.8, 70.5, 68.9,
66.5, 55.5, 39.0, 27.3; ESI MS m/z (M)þ: calcd 512.2 obsd
513.6.
8.1.11. Allyl 3-O-benzyl-6-O-p-methoxybenzyl-2-O-piva-
loyl-b-D-glucopyranoside. Allyl 3-O-benzyl-4,6-di-O-p-
methoxybenzylidene-2-O-pivaloyl-b-D-glucopyranoside 10
(609 mg, 1.19 mmol) was dissolved in DMF (8.0 mL) and
˚
4 A flame dried molecular sieves (250 mg) were added. The
resulting mixture was stirred for 30 min, NaCNBH3
(330 mg, 5.3 mmol) were added. After 30 min, the mixture
was cooled to 0 8C and trifluoroacetic acid (810 mL in 6 mL
DMF) was added dropwise. The resulting solution was
allowed to warm and stir over 12 h. The mixture was diluted
with CH2Cl2 (10 mL) and passed through a pad of celite.
The mixture was washed with sat. NaHCO3 (20 mL) and the
aqueous layers were extracted with CH2Cl2 (3£20 mL) and
the combined organic layers were dried over MgSO4,
filtered and the solvents removed in vacuo. Column
chromatography (3:1 hexanes–EtOAc) afforded allyl 3-O-
benzyl-6-O-p-methoxybenzyl-2-O-pivaloyl-b-D-glucopyra-
noside as a yellow oil (455 mg, 76%). [a]2D4¼237.88 (c
8.1.9. tert-Butyldimethylsilyl 3-O-benzyl-2-O-pivaloyl-
b-D-glucopyranoside 8. tert-Butyldimethylsilyl 3-O-ben-
zyl-4,6-di-O-benzylidene-2-O-pivaloyl-b-D-glucopyrano-

7762
E. R. Palmacci, P. H. Seeberger / Tetrahedron 60 (2004) 7755–7766
2.30, CH2Cl2); IR (thin film, NaCl) 3413, 2968, 2872, 1738,
1
water (1.0 mL). Ceric ammonium nitrate (660 mg,
1.2 mmol) was added, the mixture was stirred for 30 min
and diluted with CH2Cl2 (20 mL). The organic layer was
washed with sat. NaHCO3 (20 mL) and the aqueous layer
was extracted with CH2Cl2 (3£20 mL). The combined
organic layers were dried over MgSO4, filtered and the
solvents removed in vacuo. Column chromatography (3:2
1073 cm21; H NMR (500 MHz, CDCl3) d 7.36–7.25 (m,
7H), 6.88 (d, J¼7.8 Hz, 2H), 5.90–5.80 (m, 1H), 5.28–5.16
(m, 1H), 5.07 (app t, J¼7.9 Hz, 1H), 4.74 (d, J¼11.5 Hz,
1H), 4.70 (d, J¼11.5 Hz, 1H), 4.55 (d, J¼11.6 Hz, 1H), 4.51
(d, J¼11.6 Hz, 1H), 4.45 (d, J¼7.9 Hz, 1H), 4.35–4.31 (m,
1H), 4.05–4.01 (m, 1H), 3.81 (s, 3H), 3.75–3.73 (m, 2H),
3.56 (app t, J¼9.5 Hz, 1H), 3.50–3.47 (m, 1H), 1.20 (s, 9H);
13C NMR (125 MHz, CDCl3) d 177.0, 159.5, 138.4, 133.8,
130.0, 129.6, 128.7, 128.0, 127.8, 117.5, 114.0, 100.4, 82.8,
74.5, 74.2, 73.6, 72.7, 72.3, 70.2, 70.1, 55.5, 39.0, 27.3; ESI
MS m/z (MþNH4)þ: calcd 532.3 obsd 532.6.
hexanes–EtOAc)
afforded
12
(267 mg,
70%).
[a]2D4¼220.68 (c 0.36, CH2Cl2); IR (thin film, NaCl)
3471, 2968, 2921, 1739, 1087 cm21; H NMR (400 MHz,
1
CDCl3) d 7.32–7.22 (m, 5H), 5.90–5.80 (m, 1H), 5.30–
5.25 (m, 2H), 5.29–5.00 (m, 3H), 4.67 (d, J¼11.2 Hz, 1H),
4.56 (d, J¼11.6 Hz, 1H), 4.51 (d, J¼7.6 Hz, 1H), 4.37–4.32
(m, 1H), 4.10–4.05 (m, 1H), 3.80 (app t, J¼9.2 Hz, 1H),
3.70 (dd, J¼2.4, 12.4 Hz, 1H), 3.60 (dd, J¼5.2, 12.4 Hz,
1H), 3.45–3.40 (m, 1H), 1.97 (s, 3H), 1.21 (s, 9H); 13C
NMR (125 MHz, CDCl3) d 176.3, 170.5, 138.0, 133.7,
128.6, 127.9, 127.7, 117.8, 100.4, 80.4, 74.6, 73.9, 72.5,
70.4, 70.2, 61.8, 39.0, 27.3, 21.0; ESI MS m/z (MþNH4)þ:
calcd 454.6 obsd 454.6.
8.1.12. Allyl 3-O-benzyl-4-O-p-methoxybenzyl-2-O-piva-
loyl-b-D-glucopyranoside 11. Allyl 3-O-benzyl-4,6-di-O-
p-methoxybenzylidene-2-O-pivaloyl-b-D-glucopyranoside
10 (400 mg, 0.766 mmol) was co-evaporated with toluene
(3£5 mL) and dissolved in CH3CN (15 mL). The solution
˚
was cooled to 0 8C and 4 A flame dried molecular sieves
(200 mg) and NaCNBH3 (290 mg, 4.60 mmol) were
added. TMSCl (580 mL, 4.60 mmol, in 5 mL CH3CN)
was added and the solution was allowed to stir and warm
to room temperature. After 2 h, TMS chloride (100 mL)
was added and the solution was stirred at room
temperature for 4 h. The solution was diluted with
CH2Cl2 (20 mL) and passed through a plug of celite.
The organic layer was washed with sat. NaHCO3 and the
aqueous layer was extracted with CH2Cl2 (2£25 mL).
The combined organic layers were dried over MgSO4,
filtered and the solvents were removed in vacuo. Column
chromatography (3:1 hexanes–EtOAc) afforded a 1:1
mixture of the 6-O-PMB ether and 4-O-PMB ether 11
(259 mg, 66%). Determination of the regiochemistry was
accomplished by comparison of 1H NMR in DMSO-d6
before and after acetylation of the separate isomers. The
acetylated hydroxyl results in a downfield shift of the
ring proton that shows the expected apparent triplet for
H4 and complex splitting for the H6 methylene; IR (thin
8.1.14. Allyl 3-O-benzyl-4-O-levulinoyl-6-O-p-methoxy-
benzyl-b-D-glucopyranoside. Allyl 4-O-benzyl-6-O-p-
methoxybenzyl-2-O-pivaloyl-b-D-glucopyranoside (3.2 g,
6.44 mmol) was dissolved in CH2Cl2 (60 mL) and the
solution was cooled to 0 8C. DMAP (780 mg, 6.39 mmol),
Diisopropylcarbodiimide (DIPC) (1.6 mL, 10.2 mmol) and
levulinic acid (1.0 mL, 9.6 mmol) were added, the mixture
was shielded from light and was allowed to stir and warm
for 3 h. Ethyl acetate (100 mL) was added and the mixture
was passed through a plug of silica. The organic layer was
washed with sat. NaHCO3, dried over MgSO4, filtered and
the solvents were removed in vacuo. Column chromato-
graphy (3:1 hexanes–EtOAc) afforded allyl 3-O-benzyl-4-
O-levulinoyl-6-O-p-methoxybenzyl-b-D-glucopyranoside
(3.40 g, 87%). [a]2D4¼216.18 (c 3.45, CH2Cl2); IR (thin
1
film, NaCl) 2969, 2873, 1741, 1718, 1151 cm21; H NMR
(400 MHz, CDCl3) d 7.33–7.23 (m, 9H), 6.88–6.84 (m,
2H), 5.89–5.80 (m, 1H), 5.29–5.06 (m, 4H), 4.63 (d,
J¼11.3 Hz, 1H), 4.55 (d, J¼11.3 Hz, 1H), 4.50–4.47 (m,
3H), 4.36 (ddt, J¼1.5, 4.9, 13.0 Hz, 1H), 4.05 (ddt, J¼1.3,
6.2, 13.0 Hz, 1H), 3.80 (s, 3H), 2.67–2.50 (m, 2H), 2.44–
2.25 (m, 2H), 2.12 (s, 3H), 1.20 (s, 9H); 13C NMR
(125 MHz, CDCl3) d 206.4, 176.8, 171.7, 159.4, 138.2,
133.7, 130.3, 129.7, 128.5, 127.8, 127.7, 117.7, 113.9,
100.2, 80.5, 77.5, 73.8, 73.5, 73.4, 72.4, 70.9, 70.1, 69.4,
55.4, 38.9, 37.9, 29.9, 28.0, 27.3; ESI MS m/z (MþNH4)þ:
calcd 630.3 obsd 630.6.
film, NaCl) 3447, 2965, 2918, 1735, 1077 cm21 1H
;
NMR (400 MHz, CDCl3) d 7.35–7.27 (m, 5H), 7.19 (d,
J¼8.6 Hz, 2H), 6.86 (d, J¼8.6 Hz, 2H), 5.90–5.81 (m,
1H), 5.29–5.17 (m, 2H), 5.06 (dd, J¼8.1, 8.2 Hz, 1H),
4.79 (d, J¼11.1 Hz, 1H), 4.74 (d, J¼10.6 Hz, 1H), 4.71
(d, J¼11.1 Hz, 1H), 4.56 (d, J¼10.6 Hz, 1H), 4.47 (d,
J¼8.0 Hz, 1H), 4.32 (ddt, J¼1.3, 5.2, 13.0 Hz, 1H), 4.05
(ddt, J¼1.3, 6.0, 13.0 Hz, 1H), 3.87 (dd, J¼2.7, 12.0 Hz,
1H), 3.80 (s, 3H), 3.76–3.63 (m, 3H), 3.41–3.37 (m,
1H), 1.19 (s, 3H); 13C NMR (100 MHz, CDCl3) d 177.0,
159.6, 138.3, 133.7, 130.1, 130.0, 128.6, 127.9, 127.6,
117.7, 114.1, 100.6, 83.2, 77.4, 77.4, 75.5, 75.2, 75.0,
70.5, 62.2, 55.5, 39.0, 27.3; ESI MS m/z (MþNH4)þ:
calcd 532.21 obsd 532.6.
8.1.15. Allyl 3-O-benzyl-4-O-levulinoyl-2-O-pivaloyl-b-
D-glucopyranoside 13. Allyl 4-O-benzyl-6-O-p-methoxy-
benzyl-2-O-pivaloyl-b-D-glucopyranoside
(119 mg,
0.231 mmol) was dissolved in CH2Cl2 (3.0 mL), the
solution was cooled to 0 8C and DIPC (54.0 mL,
0.350 mmol) and DMAP (28 mg, 0.231 mmol) were
added. Levulinic acid (37.0 mL, 0.347 mmol) was added
and the reaction mixture was shielded from light and
allowed to stir for 15 h. The mixture was diluted with
EtOAc (10 mL) and the solution was passed through a plug
of silica and the solvents were removed in vacuo. The
residue was dissolved in CH3CN (900 mL) and water
(100 mL) and ceric ammonium nitrate (240 mg,
0.444 mmol) was added. After 30 min, the mixture was
8.1.13. Allyl 4-O-acetyl-4-O-benzyl-2-O-pivaloyl-b-D-
glucopyranoside 12. Allyl 4-O-benzyl-6-O-p-methoxyben-
zyl-2-pivaloyl-b-D-glucopyranoside (450 mg, 0.875 mmol)
was dissolved in CH2Cl2 (5.0 mL) and pyridine (1.0 mL).
Acetic anhydride (125 mL, 1.10 mmol) and DMAP
(0.100 g, 0.875 mmol) were added. After 30 min, the
reaction mixture was diluted with CH2Cl2 (10 mL) and
the organic layer was washed with sat. NaHCO3 (10 mL),
dried over MgSO4, filtered and the solvents were removed
in vacuo. The residue was dissolved in CH3CN (9.0 mL) and

E. R. Palmacci, P. H. Seeberger / Tetrahedron 60 (2004) 7755–7766
7763
diluted with CH2Cl2 (10 mL) and the organic layer was
washed with sat. NaHCO3 (10 mL). The aqueous layer was
extracted with CH2Cl2 (3£10 mL), the combined organic
layers were dried over MgSO4, filtered and the solvents
were removed in vacuo. Column chromatography (1:1
MgSO4, filtered and the solvents were removed in vacuo to
give the acid as a yellow oil (2.0 g, 91%). A portion of the
acid (557 mg, 1.32 mmol) was dissolved in DMF (15 mL)
and NaHCO3 (230 mg, 2.77 mmol) and MeI (90.0 mL,
1.45 mmol) were added. After 15 h, the mixture was diluted
with EtOAc (40 mL) and the organic layer was washed with
water (50 mL). The aqueous layers were extracted with
EtOAc (2£75 mL) and the combined organic layers were
dried over MgSO4, filtered and the solvents were removed
in vacuo. Column chromatography (5:1!3:2 hexanes–
EtOAc) afforded methyl (allyl 3-O-benzyl-4-O-levulinoyl-
2-O-pivaloyl-b-D-glucopyranosid)uronate (406 mg, 59%).
[a]2D4¼þ1.28 (c 0.34, CH2Cl2); IR (thin film, NaCl) 2973,
hexanes–EtOAc)
afforded
13
(89.0 mg,
85%).
[a]2D4¼235.58 (c 2.28, CH2Cl2); IR (thin film, NaCl)
3373, 2970, 2908, 1738, 1722 cm21; H NMR (500 MHz,
1
CDCl3) d 7.39–7.25 (m, 5H), 5.90–5.80 (m, 1H), 5.30–
5.06 (m, 4H), 4.66 (d, J¼11.4 Hz, 1H), 4.60 (d, J¼11.4 Hz,
1H), 4.52 (d, J¼8.1 Hz, 1H), 4.36–4.32 (m, 1H), 4.09–4.05
(m, 1H), 3.81 (app t, J¼8.8 Hz, 1H), 3.75 (dd, J¼2.6,
12.7 Hz, 1H), 3.67 (dd, J¼4.6, 12.7 Hz, 1H), 3.46–3.42 (m,
1H), 2.82–2.77 (m, 1H), 2.61–2.50 (m, 2H), 2.31–2.25 (m,
1H), 2.18 (s, 3H), 1.21 (s, 9H); 13C NMR (125 MHz,
CDCl3) d 206.7, 176.8, 138.2, 133.7, 128.5, 127.8, 127.6,
117.8, 100.3, 80.4, 74.6, 73.9, 72.6, 70.7, 70.3, 61.6, 39.0,
38.0, 29.9, 27.9, 27.3, 23.5; ESI MS m/z (MþNH4)þ: calcd
510.2 obsd 510.6.
1
1765, 1733, 1153 cm21; H NMR (500 MHz, CDCl3) d
7.38–7.25 (m, 5H), 5.88–5.80 (m, 1H), 5.29–5.15 (m, 4H),
4.66 (d, J¼11.4 Hz, 1H), 4.61 (d, J¼11.4 Hz, 1H), 4.54 (d,
J¼7.6 Hz, 1H), 4.39–4.35 (m, 1H), 4.07–4.03 (m, 1H),
3.98 (d, J¼9.8 Hz, 1H), 3.80 (app t, J¼9.2 Hz, 1H), 3.75 (s,
3H), 2.72–2.35 (m, 4H), 2.15 (s, 3H), 1.20 (s, 9H); 13C
NMR (100 MHz, CDCl3) d 206.3, 176.7, 171.6, 168.0,
137.9, 133.4, 128.5, 127.9, 127.7, 118.1, 100.0, 79.8, 73.9,
73.1, 72.1, 71.2, 70.3, 53.1, 39.0, 37.8, 30.0, 27.8, 27.3; ESI
MS m/z (MþNH4)þ: calcd 538.2 obsd 538.6.
8.1.16. Methyl (allyl 4-O-acetyl-3-O-benzyl-2-O-pivaloyl-
b-D-glucopyranosid)uronate 15. Allyl 4-O-acetyl-3-O-
benzyl-2-O-pivaloyl-b-D-glucopyranoside 12 (125 mg,
0.285 mmol) was dissolved in CH3CN (2.5 mL) and water
(18 mL) and the solution was cooled to 0 8C. A solution of
H5IO6/CrO3 (1.5 mL, 2.5 equiv./1.1 mol% in 0.75% H2O/
CH3CN) was added. After 90 min, the mixture was diluted
with EtOAc (10 mL) and the organic layer was washed with
sat. NH4Cl (10 mL). The aqueous layer was extracted with
EtOAc (3£10 mL) and the combined organic layers were
washed with water (25 mL). The organic layers were dried
with MgSO4, filter and the solvents were removed in vacuo.
The residue was dissolved in DMF (4.0 mL) and NaHCO3
(50 mg) and MeI (25 mL, 0.42 mmol) were added. After
15 h, the solution was diluted with EtOAc and the organic
layer was washed with 5% aq. HCl (10 mL) and water
(10 mL). The aqueous layer was extracted with EtOAc
(3£10 mL) and the organic layer was dried over MgSO4,
filtered and the solvents were removed in vacuo. Column
chromatography (3:1 hexanes–EtOAc) afforded 15
(90.4 mg, 70%). [a]D24¼26.38 (c 0.30, CH2Cl2); IR (thin
8.1.18. Methyl (allyl 3-O-benzyl-2-O-pivaloyl-b-D-gluco-
pyranosid)uronate 16. Methyl (allyl 3-O-benzyl-4-O-levu-
linoyl-2-O-pivaloyl-b-D-glucopyranosid)uronate (500 mg,
0.962 mmol) was dissolved in CH2Cl2 (10 mL) and
MeOH (1.0 mL) and hydrazine acetate (88 mg,
0.962 mmol) was added. After 1 h, acetone (10 mL) was
added and the solvents were removed in vacuo. Column
chromatography (3:1 hexanes–EtOAc) afforded 16 as a
colorless syrup (480 mg, 97%). [a]2D4¼231.08 (c 0.12,
CH2Cl2); IR (thin film, NaCl) 3351, 2921, 1738,
1
1731 cm21; H NMR (300 MHz, CDCl3) d 27.35–7.26
(m, 5H), 5.85–5.73 (m, 1H), 5.27–5.14 (m, 1H), 5.05 (app
t, J¼7.7 Hz, 1H), 4.76 (d, J¼11.6 Hz, 1H), 4.69 (d,
J¼11.6 Hz, 1H), 4.48 (d, J¼7.6 Hz, 1H), 4.36–4.30 (m,
1H), 4.06–3.95 (m, 3H), 3.87–3.80 (m, 2H), 3.79 (s, 3H),
3.58 (app t, J¼8.8 Hz, 1H), 2.95 (br s, 1H), 1.18 (s, 9H); 13C
NMR (125 MHz, CDCl3) d 176.9, 169.9, 138.2, 133.4,
128.7, 127.9, 127.8, 118.0, 100.5, 81.2, 74.7, 74.4, 72.3,
71.9, 70.4, 53.1, 39.0, 27.3; ESI MS m/z (MþNH4)þ: calcd
440.2 obsd 440.6.
film, NaCl) 2971, 1740, 1764, 1147 cm21 1H NMR
;
(400 MHz, CDCl3) d 7.34–7.22 (m, 5H), 5.90–5.79 (m,
1H), 5.30–5.20 (m, 4H), 4.67 (d, J¼11.6 Hz, 1H), 4.57 (d,
J¼11.6 Hz, 1H), 4.54 (d, J¼7.6 Hz, 1H), 4.40–4.35 (m,
1H), 4.08–4.03 (m, 1H), 3.95 (d, J¼9.6 Hz, 1H), 3.81–3.76
(m, 1H), 3.75 (s, 3H), 1.95 (s, 3H), 1.21 (s, 9H); 13C NMR
(100 MHz, CDCl3) d 176.7, 169.6, 168.1, 137.9, 133.4,
128.6, 127.9, 127.7, 118.1, 100.1, 79.8, 77.6, 73.8, 73.2,
72.1, 70.8, 70.3, 53.1, 39.0, 27.3, 20.8; ESI MS m/z
(MþNH4)þ: calcd 482.6 obsd 464.2.
8.1.19. Allyl (4-O-benzoyl-6-O-benzyl-2-deoxy-3-O-levu-
linoyl-2-trichloroacetimido-b-D-glucopyranosyl)-(1!4)-
(methyl 3-O-benzyl-2-O-pivaloyl-b-D-glucopyranosyl)
uronate 17. 4-O-Benzoyl-6-O-benzyl-2-deoxy-3-O-levuli-
noyl-2-trichloroacetimido-a-D-glucopyranosyl trichloro-
acetimidate 5 (70 mg, 0.093 mmol) and allyl (methyl 3-O-
benzyl-2-O-pivaloyl-b-D-glucopyranosyl)uronate 16 (30 mg,
0.071 mmol) were combined and co-evaporated with
toluene (3£5 mL) and dissolved in CH2Cl2 (1.5 mL). The
resulting solution was cooled to 0 8C and TMSOTf (1.0 mL)
was added. After 30 min, Et3N (10 mL) was added and the
solvents were removed in vacuo. Column chromatography
(3:1 hexanes–EtOAc) afforded 17 (62.4 mg, 86%); IR (thin
8.1.17. Methyl (allyl 3-O-benzyl-4-O-levulinoyl-2-O-
pivaloyl-b-D-glucopyranosid)uronate. Allyl 3-O-benzyl-
4-O-levulinoyl-2-O-pivaloyl-b-D-glucopyranoside
13
(2.20 g, 4.31 mmol) was dissolved in CH3CN (40 mL) and
water (300 mL) and the resulting solution was cooled to
0 8C. A solution of H5IO6/CrO3 (25 mL, 2.5 equiv./
1.1 mol%, 0.75% water/CH3CN) was added dropwise.
After 1.5 h, the mixture was diluted with EtOAc (60 mL)
and the organic layers were washed with sat. NH4Cl
(75 mL). The aqueous layers were extracted with EtOAc
(2£75 mL) and the combined organic layers were dried over
film, NaCl) 2957, 2872, 1726, 1270 cm21 1H NMR
;
(300 MHz, CDCl3) d 7.90 (d, J¼8.4 Hz, 2H), 7.57 (t,
J¼6.8 Hz, 1H), 7.39 (t, J¼8.0 Hz, 2H), 7.32–7.01 (M,
10H), 5.87–5.79 (m, 1H), 5.38–5.18 (M, 4H), 5.07–4.97
(m, 3H), 4.62 (d, J¼11.6 Hz, 1H), 4.51 (d, J¼6.8 Hz, 1H),

7764
E. R. Palmacci, P. H. Seeberger / Tetrahedron 60 (2004) 7755–7766
4.35–4.30 (m, 1H), 4.20–3.93 (m, 5H), 3.94 (d, J¼9.2 Hz,
1H),.3.84 (s, 3H), 3.77–3.69 (m, 2H), 3.48–3.44 (m, 1H),
3.29–3.25 (m, 1H), 2.60–2.57 (m, 2H), 2.46–2.41 (m, 2H),
2.03 (s, 3H), 1.41 (s, 9H); 13C NMR (100 MHz, CDCl3) d
205.9, 177.0, 172.7, 170.2, 165.5, 162.2, 138.8, 137.8,
133.6, 130.0, 129.3, 128.6, 128.4, 128.4, 127.8, 127.6,
127.5, 127.0, 118.1, 100.6, 100.5, 92.6, 80.7, 78.5, 77.5,
75.3, 74.2, 74.2, 73.7, 73.0, 70.5, 69.9, 69.5, 56.3, 53.3,
38.9, 38.0, 29.7, 28.2, 27.2; ESI MS m/z (MþNH4)þ: calcd
1065.4 obsd 1065.6.
8.1.22. Allyl 4-O-acetyl-6-O-benzyl-2-deoxy-3-O-levuli-
noyl-2-trichloroacetimido-b-D-glucopyranoside. Allyl
4,6-O-benzylidene-2-deoxy-3-O-levulinoyl-2-trichloroace-
timido-b-D-glucopyranoside (2.03 g, 3.69 mmol) was dis-
solved in CH2Cl2 (35 mL) and the solution was cooled to
0 8C. Triethylsilane (2.3 mL, 14.76 mmol) and trifluoroace-
tic acid anhydride (50 mL, 0.352 mmol) was added.
Trifluoroacetic acid (1.14 mL, 14.76 mmol) was added
dropwise and the solution was stirred for 1 h at 0 8C and
3 h at ambient temperature. CH2Cl2 (50 mL) and water
(50 mL) were added and the organic layer was washed with
sat. NaHCO3. The aqueous layer was extracted with CH2Cl2
(3£50 mL), the combined organic layers were dried over
MgSO4, filtered and the solvents were removed in vacuo.
The residue was dissolved in CH2Cl2 and acetic anhydride
(500 mL, 5.2 mmol) and DMAP (1.3 g, 10.7 mmol) were
added. After 1 h, the mixture was diluted with hexanes–
EtOAc (3:2, 30 mL) and the mixture was passed through a
silica plug and the solvents were removed in vacuo. Column
chromatography (3:2 hexanes–EtOAc) afforded allyl 4-O-
acetyl-6-O-benzyl-2-deoxy-3-O-levulinoyl-2-trichloroace-
timido-b-D-glucopyranoside as a yellow syrup (1.82 g,
83%). [a]2D4¼26.18 (c 7.42, CH2Cl2); IR (thin film, NaCl)
8.1.20. Allyl 4,6-di-O-benzylidene-2-deoxy-3-O-levuli-
noyl-2-trichloroacetimido-b-D-glucopyranoside. Allyl
4,6-di-O-benzylidene-2-deoxy-2-trichloroacetimido-b-D-
glucopyranoside 18 (1.56 g, 3.45 mmol) was dissolved in
CH2Cl2 (20 mL) and the solution was cooled to 0 8C. DIPC
(860 mL, 5.52 mmol) and DMAP (420 mg, 3.45 mmol)
were added followed by the addition of levulinic acid
(545 mL, 5.18 mmol). The reaction mixture was shielded
from light and was allowed to stir and warm for 13 h. Ethyl
acetate (50 mL) was added and the organic layer was
washed with sat. NaHCO3. The aqueous layer was extracted
with EtOAc (3£50 mL) and the combined organic layers
were dried over MgSO4, filtered and the solvents were
removed in vacuo. Column chromatography (5% MeOH/
CH2Cl2) afforded allyl 4,6-di-O-benzylidene-2-deoxy-
3-O-levulinoyl-2-trichloroacetimido-b-D-glucopyranoside
(1.87 g, 98%). [a]2D4¼25.08 (c 0.30, CH2Cl2); IR (thin film,
NaCl) 2966, 2933, 1746, 1716, 1615 cm21 1H NMR
;
(500 MHz, CDCl3) d 7.50–7.27 (m, 5H), 5.85–5.77 (m,
1H), 5.54–5.50 (m, 2H), 5.25–5.14 (m, 2H), 4.58 (d,
J¼8.4 Hz, 1H), 4.20 (dd, J¼5.0, 13.1 Hz, 1H), 4.15–4.09
(m, 2H), 3.91 (dd, J¼5.9, 12.1 Hz, 1H), 3.74–3.69 (m, 2H),
3.59–3.54 (m, 1H), 2.76–2.73 (m, 2H), 2.66–2.56 (m, 2H),
2.13 (s, 3H); 13C NMR (125 MHz, CDCl3) d 205.9, 173.4,
162.4, 137.2, 133.4, 129.1, 128.4, 126.1, 117.7, 101.1,
100.6, 78.9, 71.7, 70.7, 68.5, 66.1, 56.2, 38.1, 29.9, 28.3,
23.6; ESI MS m/z (MþNH4)þ: calcd 567.0 obsd 567.4.
1
2916, 2873, 1750, 1718, 1226 cm21; H NMR (500 MHz,
CDCl3) d 7.34–7.26 (m, 5H), 6.99 (d, J¼8.8 Hz, 1H), 5.86–
5.81 (m, 1H), 5.37 (app t, J¼9.6 Hz, 1H), 5.28–5.16 (m,
1H), 5.11 (app t, J¼9.8 Hz, 1H), 4.72 (d, J¼8.2 Hz, 1H),
4.57 (d, J¼11.9 Hz, 1H), 4.51 (d, J¼11.9 Hz, 1H), 4.09 (dd,
J¼6.1, 13.1 Hz, 1H), 3.99–3.93 (m, 1H), 3.73–3.70 (m,
1H), 3.59–3.54 (m, 2H), 2.72–2.67 (m, 2H), 2.55–2.40 (m,
2H), 2.13 (2, 3H), 1.98 (s, 3H); 13C NMR (125 MHz,
CDCl3) d 206.4, 172.7, 170.0, 162.1, 137.8, 133.4, 128.6,
128.1, 128.0, 118.1, 99.5, 73.8, 73.7, 72.0, 70.4, 69.0, 69.0,
56.4, 37.9, 29.8, 28.2, 20.9; ESI MS m/z (MþNH4)þ: calcd
611.1 obsd 611.4.
8.1.23. Thioethyl 2-O-benzoyl-3-O-benzyl-4-O-levuli-
noyl-6-O-p-methoxybenzyl-b-D-glucopyranoside
22.
Thioethyl 2-O-benzoyl-3-O-benzyl-4,6-di-O-p-methoxyben-
zylidene-b-D-glucopyranoside 2134 (175 mg, 0.326 mmol)
8.1.21. Allyl 6-O-benzyl-2-deoxy-3-O-levulinoyl-2-tri-
chloroacetimido-b-D-glucopyranoside 19. Allyl 4,6-di-O-
benzylidene-2-deoxy-3-O-levulinoyl-2-trichloroacetimido-
b-D-glucopyranoside (1.10 g, 2.00 mmol) was dissolved in
CH2Cl2 (20 mL) and the solution was cooled to 0 8C.
Triethylsilane (1.27 mL, 7.97 mmol) and trifluoroacetic acid
anhydride (30.0 mL, 0.211 mmol) was added followed by
the dropwise addition of trifluoroacetic acid (616 mL,
7.97 mmol). After 10 h, sat. NaHCO3 (50 mL) was added
and the aqueous layer was extracted with CH2Cl2
(3£50 mL), the organic layer was dried over MgSO4, filtered
and the solvents were removed in vacuo. Column chroma-
tography (3:1!3:2 hexanes–EtOAc) afforded 19 as a
yellow syrup (800 mg, 72%); IR (thin film, NaCl) 3342,
˚
was dissolved in DMF (3.0 mL) and 4 A flame dried
molecular sieves (100 mg) were added and the resulting
solution was stirred for 2 h. NaCNBH3 (103 mg,
1.63 mmol) was added and the mixture was cooled to
0 8C. Trifluoroacetic acid (250 mL, 3.26 mmol) was added
dropwise and the reaction mixture was allowed to warm and
stir for 14 h. Ethyl acetate (30 mL) was added and the
mixture passed through a plug of celite. The organic layer
was washed with sat. NaHCO3 and the aqueous layer was
extracted with EtOAc (3£30 mL), the organic layers were
dried over MgSO4, filtered and the solvents were removed
in vacuo. The residue was dissolved in CH2Cl2 (3 mL) and
the solution was cooled to 0 8C. DIPC (81 mL, 0.522 mmol)
and DMAP (40 mg, 0.326 mmol) were added followed by
the addition of levulinic acid (49 mL, 0.489 mmol). The
reaction mixture was shielded from light and was allowed to
stir and warm for 13 h. Ethyl acetate (10 mL) was added and
the organic layer was washed with sat. NaHCO3 (20 mL).
The aqueous layer was extracted with EtOAc (3£20 mL)
and the combined organic layers were dried over MgSO4,
filtered and the solvents were removed in vacuo. Column
chromatography (3:1 hexanes–EtOAc) afforded 22 as a
white solid (115 mg, 56%). [a]2D4¼þ32.98 (c 1.02, CH2Cl2);
1
2918, 1717, 1065 cm21; H NMR (500 MHz, CDCl3) d
7.40–7.27 (m, 4H), 6.85–6.90 (d, J¼9.0 Hz, 1H), 5.81–
5.89 (m, 4H), 5.29–5.17 (m, 2H), 4.65–4.59 (m, 2H), 4.38–
4.34 (m, 1H), 4.10–4.06 (m, 1H), 3.97 (dt, J¼8.8, 10.8 Hz,
1H), 3.84–3.77 (m, 2H), 3.60 (q, J¼4.7 Hz, 1H), 3.33 (d,
J¼3.1 Hz, 1H), 2.85–2.77 (m, 2H), 2.64–2.48 (m, 2H), 2.20
(s, 3H); 13C NMR (125 MHz, CDCl3) d 207.8, 173.6, 162.2,
138.0, 133.6, 128.7, 128.0, 127.9, 117.9, 99.8, 92.6, 77.5,
75.3, 74.6, 73.9, 70.3, 70.2, 69.9, 55.7, 38.6, 30.0, 28.4; ESI
MS m/z (MþNH4)þ: calcd 569.1 obsd 569.4.

E. R. Palmacci, P. H. Seeberger / Tetrahedron 60 (2004) 7755–7766
7765
IR (thin film, NaCl) 2930, 2870, 1720, 1514 cm21; 1H NMR
(125 MHz, CDCl3) d 8.04–8.02 (m, 2H), 7.61–7.58 (m,
1H), 7.48–7.44 (m, 2H), 7.27 (dt, J¼2.1, 5.6 Hz, 2H),
7.16–7.11 (m, 5H), 6.88 (dt, J¼2.1, 6.7 Hz, 2H), 5.33 (dd,
J¼9.3, 9.4 Hz, 1H), 5.14 (app t, J¼9.7 Hz, 1H), 4.59 (s,
1H), 4.46 (d, J¼11.3 Hz, 1H) 3.87 (t, J¼9.2 Hz, 1H), 3.81
(s, 3H), 3.69–3.58 (m 3H), 2.75–2.60 (m, 4H), 2.44–2.32
(m 2H), 2.41 (s, 3H), 1.24 (t, J¼7.5 Hz, 3H); 13C NMR
(125 MHz, CDCl3) d 206.4, 171.7, 165.2, 159.4, 137.9,
133.4, 130.3, 130.0, 129.9, 129.7, 128.6, 128.4, 128.0,
127.8, 113.9, 83.8, 81.5, 78.0, 74.3, 73.4, 72.2, 71.4, 69.6,
55.5, 37.9, 29.9, 28.0, 24.3, 15.1; ESI MS m/z (MþNH4)þ:
calcd 654.2 obsd 654.6.
8.1.25. Allyl (methyl 2-O-benzoyl-3-O-benzyl-4-O-levuli-
noyl-b-D-glucopyranosyl)-(1!3)-4-O-acetyl-6-O-benzyl-
2-deoxy-2-trichloroacetimido-b-D-glucopyranoside 24.
Allyl (2-O-benzoyl-3-O-benzyl-4-O-levulinoyl-b-D-glu-
copyranosyl)-(1!3)-4-O-acetyl-6-O-benzyl-2-deoxy-2-tri-
chloroacetimido-b-D-glucopyranoside
23
(30.7 mg,
0.0315 mmol) was dissolved in CH3CN (1.0 mL) and
water (7 mL) was added. The solution was cooled to 0 8C
and a solution of H5IO6/CrO3 (180 mL, 2.5 equiv/1.1 mol%
in 0.75% H2O/CH3CN) was added portion-wise over
30 min. The resulting solution was stirred at 0 8C for
45 min and aqueous Na2HPO4 (1 mL) was added. CH2Cl2
(10 mL) was added and the organic layer was washed with
water (10 mL). The aqueous layer was extracted with
CH2Cl2 (3£10 mL), the combined organic layers were
washed with sat. NaHSO3, dried over MgSO4, filtered and
the solvents were removed in vacuo. The residue was
dissolved in DMF (1.0 mL) and NaHCO3 (5 mg) and
iodomethane (4 mL) were added. After 16 h, EtOAc
(5 mL) was added and the organic layer was washed with
water (5 mL). The aqueous layer was extracted with EtOAc
(3£5 mL) and the combined organic layers were dried over
MgSO4, filtered and the solvents removed in vacuo. Column
chromatography (3:2 hexanes–EtOAc) afforded 24
(12.0 mg, 39%). [a]D24¼þ40.08 (c 0.10, CH2Cl2); IR (thin
8.1.24. Allyl (2-O-benzoyl-3-O-benzyl-4-O-levulinoyl-b-
D-glucopyranosyl)-(1!3)-4-O-acetyl-6-O-benzyl-2-deoxy-
2-trichloroacetimido-b-D-glucopyranoside 23. Allyl
4-O-acetyl-6-O-benzyl-2-deoxy-3-O-levulinoyl-2-trichloro-
acetimido-b-D-glucopyranoside (100 mg, 0.168 mmol) was
dissolved in MeOH (500 mL) and CH2Cl2 (1.0 mL) and
hydrazine acetate (17 mg, 0.170 mmol) was added. After
1 h, acetone (5 mL) was added and the solvents were
removed in vacuo. Column chromatography (3:1!3:2
hexanes–EtOAc) afforded allyl 4-O-acetyl-6-O-benzyl-2-
deoxy-2-trichloroacetimido-b-D-glucopyranoside 20 (74 mg,
1
1
89%); H NMR (300 MHz, CDCl3) d 7.35–7.26 (m, 4H),
film, NaCl) 2917, 1764, 1738, 1720 1070 cm21; H NMR
7.02 (d, J¼6.8 Hz, 1H), 5.94–5.80 (m, 1H), 5.33–5.18 (m,
2H), 4.93 (app t, J¼9.2 Hz, 1H), 4.87 (d, J¼8.1 Hz, 1H),
4.61 (d, J¼12.1 Hz, 1H), 4.50 (d, J¼12.1 Hz, 1H), 4.40–
4.34 (m, 1H), 4.22–4.07 (m, 2H), 3.68–3.48 (m, 4H), 3.2
(br s, 1H), 2.00 (s, 3H). Thioethyl 2-O-benzoyl-3-O-benzyl-
4-O-levulinoyl-6-O-p-methoxybenzyl-b-D-glucopyranoside
22(77.5 mg, 0.121 mmol)and 20(40.5 mg, 0.60 mmol) were
(500 MHz, CDCl3) d 8.01 (d, J¼7.2 Hz, 2H), 7.59 (t,
J¼7.3 Hz, 1H), 7.45 (t, J¼7.9 Hz, 2H), 7.34–7.08 (m,
10H), 5.82–5.75 (m, 1H), 5.28 (dd, J¼7.7, 7.8 Hz, 1H),
5.23–5.14 (m, 1H), 5.08 (d, J¼7.9 Hz, 1H), 5.02 (app t,
J¼9.3 Hz, 1H), 4.78 (d, J¼7.6 Hz, 1H), 4.63 (d, J¼9.9 Hz,
1H), 4.58 (d, J¼13.4 Hz, 1H), 4.53 (d, J¼11.9 Hz, 1H), 4.49
(d, J¼12.1 Hz, 1H), 4.25 (dd, J¼5.2, 12.7 Hz, 1H), 4.04 (dd,
J¼6.3, 12.8 Hz, 1H), 3.95 (d, J¼9.9 Hz, 1H), 3.79 (app t,
J¼9.2 Hz, 1H), 3.72 (s, 3H), 3.64–3.61 (m, 1H), 3.54 (dd,
J¼2.9, 10.8 Hz, 1H), 3.48 (dd, J¼5.3, 10.8 Hz, 1H), 3.26–
3.23 (m, 1H), 2.73–2.39 (m, 4H), 2.17 (s, 3H), 1.74 (s, 3H);
13C NMR (100 MHz, CDCl3) d 206.0, 171.6, 170.0, 167.6,
164.6, 162.2, 138.0, 137.5, 133.6, 133.6, 130.2, 129.6,
128.7, 128.6, 128.5, 128.2, 128.1, 128.0, 127.9, 98.8, 97.6,
79.3, 77.5, 74.2, 73.8, 73.6, 72.8, 72.4, 71.5, 70.7, 69.3,
59.4, 53.1, 37.8, 30.0, 27.9, 20.6; ESI MS m/z (MþNH4)þ:
calcd 995.2 obsd 995.4.
˚
combined and dissolved in CH2Cl2 (1.0 mL) and 4 A flame
dried molecular sieves (150 mg) were added. After stirring
for 1 h, the solution was cooled to 225 8C and MeOTf
(50.0 mL, 0.400 mmol) was added. The reaction mixture
was allowed to warm to ambient temperature over 14 h.
Et3N (100 mL) was added, the mixture filtered through
celite and the solvents removed in vacuo. The residue was
dissolved in CH3CN (900 mL) and water (100 mL) and ceric
ammonium nitrate (130 mg, 0.240 mmol) was added. After
30 min, dichloromethane (10 mL) was added and the
organic layer was washed with water. The aqueous layer
was extracted with CH2Cl2 (3£10 mL), dried over MgSO4,
filtered and the solvents were removed in vacuo. Column
chromatography (3:2 hexanes–EtOAc) afforded 23
(38.1 mg, 50%). [a]D24¼þ5.38 (c 0.30, CH2Cl2); IR (thin
Acknowledgements
Financial support from the National Institute of Health (HL-
64799), the Human Frontiers Science Program (HFSP
RGY72-2002), and the DuPont-MIT Alliance is gratefully
acknowledged. Funding for the MIT-DCIF Varian 500 MHz
NMR was provided by NSF (Award CHE-9808061).
Funding for the MIT-DCIF Varian 300 MHz NMR was
provided by NSF (Award DBI-9729592).
1
film, NaCl) 3392, 2918, 1733, 1716, 1069 cm21; H NMR
(500 MHz, CDCl3) d 8.01 (d, J¼7.2 Hz, 2H), 7.59 (t,
J¼7.5 Hz, 1H), 7.46–7.43 (m, 2H), 7.35–7.08 (m, 10H),
6.99 (d, J¼7.5 Hz, 1H), 5.76–5.69 (m, 1H), 5.27–5.11 (m,
3H), 5.04–4.93 (m, 2H), 4.85 (d, J¼7.5 Hz, 1H), 4.73 (d,
J¼7.8 Hz, 1H), 4.62–4.47 (m, 5H), 4.22 (dd, J¼5.2,
12.8 Hz, 1H), 4.00 (dd, J¼6.4, 12.8 Hz, 1H), 3.80 (app t,
J¼9.2 Hz, 1H), 3.74–3.43 (m, 5H), 2.90 (br s, 1H), 2.95–
2.35 (m, 4H), 2.04 (s, 3H), 1.79 (s, 3H); 13C NMR
(125 MHz, CDCl3) d 206.6, 172.7, 170.2, 164.9, 162.1,
137.9, 137.7, 133.6, 133.4, 130.2, 130.1, 129.6, 128.7,
128.7, 128.6, 128.4, 128.3, 128.1, 128.0, 127.9, 127.9,
118.4, 99.5, 97.8, 92.5, 79.7, 75.0, 74.9, 74.2, 73.7, 73.7,
73.4, 73.3, 71.0, 70.3, 69.4, 69.2, 61.5, 57.9, 38.0, 29.9,
28.0, 20.9; ESI MS m/z (MþNH4)þ: calcd 967.2 obsd 967.4.
References and notes
1. Iozzo, R. V. Annu. Rev. Biochem. 1998, 67, 609.
¨
2. Bernfield, M.; Gotte, M.; Park, P. W.; Reizes, O.; Fitsgerald,
M. L.; Lincecum, J.; Zako, M. Annu. Rev. Biochem. 1999, 68,
729.

7766
E. R. Palmacci, P. H. Seeberger / Tetrahedron 60 (2004) 7755–7766
´
3. Roden, L.; Horowitz, M. I. Structure and Biosynthesis of
Connective Tissue Proteoglycans; Academic: New York,
1978; Vol. 2.
22. Palmacci, E. R.; Plante, O. J.; Hewitt, M. C.; Seeberger, P. H.
Helv. Chim. Acta 2003, 86, 3975.
23. Coutant, C.; Jacquinet, J.-C. J. Chem. Soc., Perkin Trans. 2
1995, 1573.
4. Mahoney, D. J.; Blundell, C. D.; Day, A. J. J. Biol. Chem.
2001, 276, 22764.
24. Blatter, G.; Jacquinet, J.-C. Carbohydr. Res. 1996, 288, 109.
25. Blatter, G.; Beau, J.-M.; Jacquinet, J.-C. Carbohydr. Res.
1994, 260, 189.
5. McKee, C. M.; Penno, M. B.; Cowman, M.; Burdick, M. D.;
Strieter, R. M.; Bao, C.; Noble, P. W. J. Clin. Invest. 1996, 98,
2403.
26. Melvin, F.; McNeill, A.; Henderson, P. J. F.; Herbert, R. B.
Tetrahedron Lett. 1999, 40, 1201.
6. Kovensky, J.; Duchaussoy, P.; Bono, F.; Salmivirta, M.;
Sizun, P.; Herbert, R. B.; Petitou, P.; Sinay¨, P. Bioorg. Med.
Chem. 1999, 7, 1567.
27. Davis, N. J.; Flitsch, S. L. Tetrahedron Lett. 1993, 34, 1181.
28. Yoshimura, J.; Sato, T.; Ando, H. Bull. Chem. Soc. Jpn 1969,
42, 2352.
7. Petitou, P.; Duchaussoy, P.; Jaurand, G.; Gourvenec, F.;
29. Wyss, P. C.; Kiss, J.; Arnold, W. Helv. Chim. Acta 1975, 58,
1847.
ˆ
´
´
Lederman, I.; Strassel, J.-M.; Barzu, T.; Crepon, B.; Herault,
J.-P.; Lormeau, J.-C.; Bernat, A.; Herbert, R. B. J. Med. Chem.
1997, 40, 1600.
30. Okuda, S.; Kondo, T.; Murata, S. Carbohydr. Res. 1990, 198,
133.
8. Yerushalmi, N.; Arad, A.; Margalit, R. Arch. Biochem.
Biophys. 1994, 313, 267.
9. Knudson, W. Front. Biosci. 1998, 3, 604.
31. Fukase, K.; Matsumoto, T.; Ito, N.; Yoshimura, T.; Kotani, S.;
Kusumoto, S. Bull. Chem. Soc. Jpn 1992, 65, 2643.
32. Herscovici, J.; Antonakis, K. J. Chem. Soc., Chem. Commun.
1980, 561.
10. Hasegawa, M.; Crowther, R. A.; Jakes, R.; Goedert, M. J. Biol.
Chem. 1997, 272, 33118.
33. Ferla, B.; Lay, L.; Guerrini, M.; Poletti, L.; Panza, L.; Russo,
G. Tetrahedron 1999, 55, 9867.
´
34. Frechet, J. M. J.; Warnock, J.; Farrall, M. J. J. Org. Chem.
1978, 43, 2618.
11. Inerot, S.; Heinegard, D. Articular Cartilage Proteoglycans in
Aging and Osteoarthritis; Academic: New York, 1982; Vol. 4.
12. Bourguignon, L. Y. W.; Zhu, H.; Shao, L.; Chen, Y. W. J. Biol.
Chem. 2000, 275, 1829.
35. Brunelet, T.; Jouitteau, C.; Gelbard, G. J. Org. Chem. 1986,
51, 4016.
13. Abatangelo, L.; Weigel, P. New Frontiers in Medical Sciences:
Redefining Hyaluronan. Elsevier: Amsterdam, 2000.
´ ´
14. Lapcık, L. Jr.; Lapcık, L.; De Smedt, S.; Demeester, J.;
36. Vermeer, H. J.; Halkes, K. M.; Albert, J.; van Kuik, A.;
Kamerling, J. P.; Vliegenthart, J. F. G. J. Chem. Soc., Perkin
Trans. 2000, 2249.
Chabrecek, P. Chem. Rev. 1998, 98, 2663.
15. DeAngelis, P. L. Glycobiology 2002, 12, 9R.
16. Blatter, G.; Jacquinet, J.-C. Carbohydr. Res. 1996, 288, 109.
17. Jacquinet, J.-C.; Rochepeau-Jbron, L.; Combal, J.-P. Carbo-
hydr. Res. 1998, 314, 283.
37. Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1990, 205, 147.
38. Lenz, R.; Ley, S. V. J. Chem. Soc., Perkin Trans. 1997, 3291.
39. Kanemoto, S.; Matsubara, S.; Takai, K.; Oshima, K. Bull.
Chem. Soc. Jpn 1988, 61, 3607.
18. Tamura, J.; Neumann, K. W.; Kurono, S.; Ogawa, T.
Carbohydr. Res. 1998, 305, 43.
40. Bandgar, B. P.; Sadavarte, V. S.; Uppalla, L. S. J. Chem. Soc.,
Perkin Trans. 2000, 3559.
19. Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem. Biochem.
1994, 50, 21.
41. Zhao, M.; Li, J.; Song, Z.; Desmond, R.; Tschaen, D. M.;
Grabowski, E. J. J.; Reider, P. J. Tetrahedron Lett. 1998, 39,
5323.
20. Plante, O. J.; Palmacci, E. R.; Seeberger, P. H. Science 2001,
291, 1523.
42. Fukase, K.; Liu, W.-C.; Suda, Y.; Oikawa, M.; Wada, A.
Tetrahedron Lett. 1995, 36, 7455.
21. Orgueira, H. A.; Bartolozzi, A.; Schell, P. H.; Litjens, R.;
Palmacci, E. R.; Seeberger, P. H. Chem. Eur. J. 2003, 9,
140–169.
43. Nilsson, M.; Svahn, C.-M.; Westman, J. Carbohydr. Res.
1993, 246, 161.