Sulfoform generation from an orthogonally protected disaccharide

Article abstract of DOI:10.1016/j.carres.2012.04.010

An orthogonally protected disaccharide (GlcN(α1→4)Glc) with a β-linked 2′-aminoethyl linker was used to generate a series of sulfated derivatives (sulfoforms), with a 6-O-sulfate on the glucose residue and one or more sulfate esters on the terminal glucosamine. Deprotection and sulfonation steps were performed in solution and in variable order, with isolated yields of 36-54% (85-90% per operation) after HPLC purification. The modular deprotection-sulfonation sequences can be performed with efficient recovery of the polysulfate products, and avoids complications associated with heterogeneous reactivity in solid-phase synthesis.

Full text of DOI:10.1016/j.carres.2012.04.010

Carbohydrate Research 355 (2012) 19–27
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Sulfoform generation from an orthogonally protected disaccharide
⇑
Runhui Liu, Oscar Morales-Collazo, Alexander Wei
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907-2084, USA
a r t i c l e i n f o
a b s t r a c t
An orthogonally protected disaccharide (GlcN(a
1?4)Glc) with a b-linked 20-aminoethyl linker was used
Article history:
Received 24 February 2012
Received in revised form 12 April 2012
Accepted 14 April 2012
to generate a series of sulfated derivatives (sulfoforms), with a 6-O-sulfate on the glucose residue and one
or more sulfate esters on the terminal glucosamine. Deprotection and sulfonation steps were performed
in solution and in variable order, with isolated yields of 36–54% (85–90% per operation) after HPLC puri-
fication. The modular deprotection–sulfonation sequences can be performed with efficient recovery of
the polysulfate products, and avoids complications associated with heterogeneous reactivity in solid-
phase synthesis.
Available online 21 April 2012
Keywords:
Sulfated carbohydrates
Microwave-assisted synthesis
Protecting groups
Glycosaminoglycans
Heparan sulfate
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
ing groups for five hydroxyls and the C2 amine of a heparan disac-
charide were previously identified and validated, and their cleavage
Proteoglycans and sulfomucins provide a number of protective
and recognition functions, the latter being involved in cell adhesion,
migration, proliferation, and infection.1,2 Heparan sulfate (HS) pro-
teoglycans and other members of this diverse family of cell-surface
carbohydrates are implicated in the recruitment of growth factors
and chemokines known to promote or inhibit angiogenesis,3,4 and
in the attraction of leukocytes that regulate the assembly and deg-
radation of the extracellular matrix.5,6 Many studies have shown
that the specificity of HS-binding proteins is defined in large part
by their affinity to specific sulfate patterns (sulfoforms) encoded
within proteoglycans,7,8 and the identification of such structures
can be applied toward the development of new anti-inflammatory
agents and immunotherapies based on glycan recognition.9,10 Even
relatively simple sulfoforms can exhibit significant influence over
protein recognition and signaling pathways,11,12 but progress in
the structure–activity relationships of HS-like compounds is ham-
pered by the difficulty of obtaining well-defined sulfoforms in suf-
ficient quantities for further study.
was also shown to be compatible with neighboring sulfate esters.13
To obtain fully deprotected sulfoforms, we have conducted modular
deblocking-sulfonation conditions on orthogonally protected glu-
cosamines (GlcNs) immobilized on tritylated polystyrene (PS) res-
ins.14,15 The solid-phase method can generate entire sets of
mono- and disaccharide sulfoforms from a common precursor with
up to three sulfate esters per GlcN, followed by mild cleavage and
ion-exchange conditions to yield the final sulfoforms as sodium
salts. We recently optimized this procedure to generate free GlcN
sulfoforms in 8 operations or less with overall yields ranging from
41% (average yield per step: 90%) to 76% (average yield per step:
>96%).15 This strategy can also produce sulfoforms with 2-amino-
ethyl linkers, to enable their conjugation onto activated substrates
or for the preparation of neoglycolipids or neoglycoproteins.
The solid-phase method of generating sulfoforms does have
some limitations, however. The mild condition for cleaving the
aminoethyl group from the trityl-PS resin (30% hexafluoroisopro-
panol in CH2Cl2) precludes the use of stronger acids in the orthog-
onal deprotection scheme. Furthermore, the efficiency of
sulfonation can vary depending on the number of sulfate esters
per glycan: as the charge density increases the environment within
the resin becomes heterogeneous, encouraging the formation of io-
nic bridges within a nonpolar environment. This can have adverse
effects on resin swelling and raise the barrier to chemical diffusion
and exchange. Resins are also mechanically fragile and may be less
well suited for reactions involving large temperature increases
(due to thermal expansion of the resin), or aggressive chemical
reagents that may cause polymer degradation.
In order to create libraries of sulfoforms with structures similar
to those found in cell-surface glycans, we have investigated the
modular deblocking and sulfonation of orthogonally protected car-
bohydrate derivatives. This approach enables us to generate both
natural and unnatural (or unidentified) sulfoforms, as opposed to
the more conventional strategy of developing precursors with
pre-designated sites for sulfonation. A set of six orthogonal protect-
⇑
Corresponding author.
E-mail address: alexwei@purdue.edu (A. Wei).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.04.010

20
R. Liu et al. / Carbohydrate Research 355 (2012) 19–27
OTBDPS
O
OAc
OTBDPS
O
O
O
STol
N3
BnO
N3
1
SEMO
PMBO
AcO
OAc
BnO
N3
O
O
OAc
3
OAc
O
BnO
NHCbz
6
OBn
a, b
d
R1
O
OPMB
O
OTBDPS
O
O
O
STol
N3
R
HO
R2
2a g
O
-O3SO
BnO
R3NH
HO
OBn
O
O
OSEM
OBn
O
HO
NH2
7
4
: R=N3
OH
c
5
: R=NHCbz
Figure 1. GlcN(
a
1?4)Glc sulfoforms with 2-aminoethyl linker (2a–g), generated
from an orthogonally protected disaccharide (1). R1,R2 = H or SO3ꢀ; R3 = Ac or SO3
ꢀ
e
(see Table 1).
OTBDPS
O
In light of these factors, we considered whether modular
deblocking-sulfonation sequences might generate fully deprotec-
ted sulfoforms in solution with comparable yields and purity as
those obtained using solid-phase methods. Working with charged
intermediates in solution can be daunting: Most synthetic routes
to sulfated oligosaccharides are designed so that sulfonation is
the last step, which minimizes the number of operations needed
to isolate these highly polar products. If the order of deprotection
and sulfonation is variable, then some charged intermediates will
be subjected to multiple processing steps. On the other hand, solu-
tion-based methods are clearly compatible with the formation of
multiple sulfate esters, so long as the products are sufficiently sta-
ble to subsequent deprotection steps.
In this article we describe seven sulfoforms generated from a
single, orthogonally protected disaccharide with a b-aminoethyl
linker (Fig. 1), using modular deblocking-sulfonation sequences
performed in solution. We focus on derivatives with a 6-O-sulfate
ester on the reducing-end glucoside and variable sulfate patterns
on the terminal glucosamine, with the latter representing the
structural diversity of HS and sulfomucins. Low molecular-weight
disaccharide sulfoforms (unnatural derivatives as well as natural)
have been found to exhibit significant binding affinity for mucin-
and HS-binding proteins such as L-selectin16,17 and FGF-2.11 We
compare the efficiency of generating sulfoforms using solution-
and solid-phase methods, and the relative benefits of reaction opti-
mization in solution versus the expedient purification of synthetic
intermediates immobilized on resins.
BnO
SEMO
PMBO
N3
O
O
O
BnO
NHCbz
OBn
1
Scheme 1. Synthesis of orthogonally protected GlcN(a1?4)Glc derivative 1.
Reaction conditions: (a) (i) NaOMe, MeOH, rt; (ii) p-MeO(C6H4)-
CH(OMe)2, CSA, THF, reflux; (iii) BnBr, NaH, DMF, 0 °C to rt (62%
over 3 steps). (b) BH3, Bu2BOTf, THF, ꢀ78 °C (99%). (c) (i) Bu3P,
CH2Cl2, then 1:1 CH2Cl2/H2O, rt; (ii) Cbz-Cl, NaHCO3, 5:1 THF/
H2O, 0 °C to rt (92% over 2 steps); (d) (i) NaOMe, MeOH, rt; (ii)
SEM-Cl, iPr2NEt, TBAI, CH2Cl2, rt (91% over 2 steps). (e) NIS, TfOH,
2:1 Et2O/(CH2Cl)2, ꢀ30 °C (68%). Selected abbreviations:
Cbz = carboxybenzyl;
PMB = p-methoxybenzyl;
methylsilyl)ethoxymethyl; TBDPS = tert-butyldiphenylsilyl; Tol =
p-tolyl.
SEM = 2-(tri-
2.2. Generation of GlcN sulfoforms
We have previously developed deblocking conditions for the
azide, PMB, SEM, and TBDPS groups that are fully orthogonal and
also compatible with sulfate esters. In this study, all derivatives
have a sulfate ester at the C6 position, so the first step is to remove
the PMB ether from 1 using ceric ammonium nitrate (CAN) in
aqueous CH3CN at 0 °C to afford C6 alcohol 8 in 88% yield (Table 1,
13
2. Results and discussion
step a). To obtain variable sulfate patterns on the 1,4-a-linked GlcN
moiety, the following operations were performed in variable order:
(i) TBDPS deprotection at the C60 position using tetrabutylammo-
nium fluoride (TBAF, step b or c), (ii) conversion of the azide at
the C20 position to –NHAc (step d)22 or a free amine (step i),23
(iii) removal of the SEM ether at the C30 position using MgBr2
and CH3NO2 (step g or h),24 and (iv) sulfonation of the free hydrox-
yls and amines (step e, f, or j). These are followed by ion-exchange
and HPLC chromatography to obtain partially benzylated sulfo-
forms 9a–g as sodium salts (step k). Finally, global deprotection
of the benzyl (Bn) and Cbz groups was performed under standard
hydrogenation conditions to obtain aminoethyl sulfoforms 2a–g
in high yields. The sequence of operations and isolated yields for
each sulfoform are summarized in Table 1.
With respect to TBDPS and SEM deprotections, there exist sig-
nificant differences in deblocking conditions when performed be-
fore or after sulfonation. Deprotections prior to sulfonation (steps
b and g) took less time (6–10 hours) than those performed after
sulfonation (steps c and h: 1–2 days); the latter also required more
polar solvents. TBDPS deprotections after sulfonation using TBAF
2.1. Synthesis of orthogonally protected GlcN(
disaccharide
a1?4)Glc
20-Azidoethyl b-glucoside tetraacetate 3, which was prepared
according to literature procedures,15,18 was converted into glycosyl
acceptor 4 in 4 steps and 61% overall yield. The azide was then con-
verted into carboxybenzyl (Cbz) carbamate
5 in 92% yield
(Scheme 1). For the reductive cleavage of the 4,6-anisylidene acetal
using BH3/Bu2BOTf, we note that the regioselectivity is tempera-
ture-dependent, and that performing the reaction at ꢀ78 °C yields
exclusively the 6-O-PMB ether.19 Orthogonally protected 2-azido-
2-deoxyglucose 6, which was also prepared according to the liter-
ature procedures,14,15 was converted into 3-O-SEM ether 7 in 91%
yield, then coupled with 5 using N-iodosuccinimide (NIS)/TfOH
activation to yield a-1,4-linked disaccharide 1 as the sole product
in 68% yield. We also examined activation conditions using ben-
zenesulfinyl piperidine (BSP)/Tf2O13,20 and phenylsulfenyl chloride
(PhSCl)/AgOTf,21 but these were found to be less effective than NIS/
TfOH activation.

R. Liu et al. / Carbohydrate Research 355 (2012) 19–27
21
Table 1
Generation of partially and fully deprotected GlcN(
a
1?4)Glc sulfoforms
OTBDPS
O
BnO
SEMO
OH
a
b
j (4-5 steps),
H2, Pd(OH)2
N3
9a
g
1
g
2a
8
O
k (ion exchange)
50% aq MeOH
O
BnO
O
NHCbz
OBn
Sulfoforma
9 (Yield from 1, ops)b
2 (Yield from 9)c
-
OSO3
O
RO
HO
-
52%, 6 ops
(a, b, d, e, h, k)
OSO3
a
b
c
d
87%
AcHN
O
O
RO
O
NHR'
NHR'
NHR'
RO
OH
O
RO
-O3SO
-
OSO3
39%, 6 ops
(a, g, d, e, c, k)
83%
84%
97%
AcHN
O
O
RO
O
RO
OH
O
RO
HO
-
OSO3
36%, 7 ops
(a, e, h, c, i, j, k)
-O3SHN
O
O
O
RO
RO
-
OSO3
O
RO
-O3SO
-
48%, 6 ops
(a, b, g, d, f, k)
OSO3
AcHN
O
O
O
RO
NHR'
RO
-
OSO3
O
RO
HO
-
42%, 6 ops
(a, b, i, f, h, k)
OSO3
e
f
98%
97%
99%
-O3SHN
O
O
RO
O
NHR'
NHR'
RO
OH
O
RO
-O3SO
-
OSO3
40%, 6 ops
(a, g, i, f, c, k)
-O3SHN
O
O
O
RO
RO
-
OSO3
O
RO
-O3SO
-
54%, 6 ops
(a, b, g, i, f, k)
OSO3
g
-O3SHN
O
O
RO
O
NHR'
RO
a
9a–g: R = Bn, R0 = Cbz; 2a–g: R, R0 = H. All products are isolated as sodium salts.
Isolated yield after HPLC purification. Each step was conducted at rt unless otherwise noted. Reagents and conditions: (a) CAN (3 equiv), 90% aq CH3CN, 0 °C, 10 h; (b)
b
TBAF (6 equiv), THF, 6 h; (c) TBAF (8–10 equiv), 1:1 THF/DMF, 24–40 h; (d) 1:5 AcSH/Py, 48 h; (e) SO3ꢁMe3N (20–30 equiv), DMF, 55 °C, 12 h; (f) SO3ꢁPy (20–30 equiv), 1:5
Et3N/Py, 70 °C, microwave, 1 h; (g) MgBr2ꢁEt2O (10 equiv), CH3NO2 (20 equiv), Et2O, 10 h; (h) MgBr2ꢁEt2O (10 equiv), 1:2 CH3NO2/Et2O, 24–48 h; (i) HS(CH2)3SH (35 equiv),
Et3N (35 equiv), MeOH, 24 h; (j) SO3ꢁPy (10 equiv), 5:1 H2O/DMF, pH 9.5, 8 h; (k) Na+ ion-exchange chromatography.
c
10% Pd(OH)2, H2 (1 atm), 1:1 MeOH/H2O, 20 h.

22
R. Liu et al. / Carbohydrate Research 355 (2012) 19–27
were also somewhat lower yielding, possibly due to complications
caused by counterion exchange with nearby sulfate esters. With
respect to the efficiency of sulfonation, we observed this reaction
to become increasingly sluggish when attempting to install several
sulfate esters under thermal conditions. We thus investigated con-
ditions involving microwave-assisted heating, which has been
3. Experimental details
3.1. General methods
All starting materials and reagents were obtained from com-
mercial sources and used as received unless otherwise noted. All
solvents were freshly distilled prior to use. IR spectra were ac-
quired from NaCl plate, using a Thermo-Nicolet Nexus 670 FT-IR
spectrometer. 1H and 13C NMR chemical shifts were referenced to
the solvent used (d 7.27 and 77.00 for CDCl3, d 3.31 and 49.15 for
CD3OD, and d 4.80 for D2O at 295 K). Mass spectra were acquired
using either a Hewlett-Packard 5989B or a Finnigan 40000 mass
spectrometer. Optical rotations were measured by polarimetry at
rt. Silica gel chromatography was performed with ICN SiliTech
32-63D. Ion-exchange chromatography was performed by dissolv-
ing sulfoforms in 50% aq MeOH (up to 100 mg/mL) and passing
them through a Dowex Marathon MSC column (14 ꢂ 1.5 cm),
which was activated with 0.1 M NaOH and washed with water
prior to use. HPLC purifications were performed by dissolving sul-
foforms in CH3CN (up to 20 mg/mL) and injecting them onto a C18
used to generate carbohydrates with multiple sulfate esters.25
A
systematic variation in reaction solvent, time, and temperature re-
vealed that microwave-assisted sulfonation is indeed more effec-
tive, but also highly temperature-sensitive. Reactions could be
performed at 70 °C with minimal decomposition and generation
of by-products, but reactions over 80 °C led to the rapid formation
of a black tar. Sulfonation was most efficient when performed in
pyridine buffered with Et3N, which also enabled simultaneous N-
and O-sulfate generation (2e–g). In comparison, the synthesis of
20,6-di-N,O-sulfate (2c) under conventional thermal conditions re-
quired separate sulfonation steps.
It is worthwhile to compare the synthetic efficiency of the mod-
ular deprotection–sulfonation sequence in solution with that per-
formed on resin supports. We recently reported the solid-phase
synthesis of a similar series of
a-glucosamine sulfoforms (mono-
reverse-phase column (250 ꢂ 10 mm, 4
lm beads, 80 Å pores)
with UV detection at 214 nm, using a binary solvent gradient (up
and disaccharides 10 and 11), with overall yields ranging from
42% to 76% over 7–8 operations (Table 2).15 These yields are higher
on average than those reported in Table 1, but not uniformly so; for
example, the solid-phase syntheses of 2,6-di-N,O-sulfate deriva-
tives 10e and 11e are comparable to the solution synthesis of
the 20,60,6-tri-N,O,O-sulfate derivative 9e. In addition, we have
encountered several practical limits with solid-phase methodolo-
gies. These limits included scalability and compound loading, the
range of solvents that permit appreciable resin swelling, and the
long reaction times required for the completion of each step, par-
ticularly sulfonation. We considered using microwave-assisted
heating to increase the efficiency of solid-phase sulfonation; how-
ever, attempts to do so with SO3ꢁMe3N in DMF at 70 °C resulted in
extensive degradation of the supporting resin.
In closing, we find that orthogonal deprotection–sulfonation se-
quences can be performed in solution without a serious attrition in
synthetic efficiency, relative to that performed by solid-phase syn-
thesis. This process can produce aminoethyl-linked disaccharide
sulfoforms in good overall yield, for subsequent conjugation onto
substrates for affinity screening against heparin-binding proteins
and pathogens.
to 40% aq CH3CN) with a flow rate of 5 mL/min.
3.2. 20-Azidoethyl 2,3-di-O-benzyl-6-O-p-methoxybenzyl-b-
D-
glucopyranoside (4)
20-Azidoethyl tetra-O-acetyl-b- -glucopyranoside 315,18 (14.2 g,
D
34.2 mmol) was dissolved in anhydrous MeOH (200 mL) and trea-
ted at rt with 1 M NaOMe solution in MeOH (10.3 mL). The mixture
was stirred at rt for 5 h, neutralized with acid-washed Dowex
50W-X8 ion-exchange resin, filtered, concentrated, and dried un-
der reduced pressure. The crude tetraol was redissolved in THF
(80 mL) and treated with p-methoxybenzaldehyde dimethyl acetal
(9.8 mL, 57.4 mmol) and camphorsulfonic acid (CSA, 476 mg,
2.1 mmol), then heated to reflux for 10 h. The reaction mixture
was quenched with saturated aq NaHCO3 (50 mL), extracted with
CH2Cl2 (3 ꢂ 150 mL), dried over Na2SO4 and concentrated, then
purified by silica gel chromatography (50% EtOAc in hexanes) to af-
ford the 4,6-anisylidene acetal as an amorphous white solid (9.3 g).
The resulting 2,3-diol was redissolved in anhydrous DMF (160 mL)
and treated at 0 °C with BnBr (5.8 mL, 49.1 mmol) and NaH (1.47 g,
Table 2
Mono- and disaccharide
a
-GlcN sulfoforms prepared by solid-phase synthesis15 (overall yields)
-
-
-
OSO3
OSO3
OSO3
10a
(76%)
10d
10g
O
O
O
BnO
BnO
BnO
(68%)
(64%)
-O3SO
-O3SO
HO
AcHN
O
AcHN
-O3SHN
O
O
NH2
NH2
NH2
-
-
OH
OSO3
OSO3
10b
10e
O
11a
O
O
BnO
BnO
HO
BnO
HO
(64%)
(44%)
-O3SO
AcHN
O
OBn
O
(69%)
-O3SHN
AcHN
O
O
O
NH2
O
NH2
BnO
NH2
BnO
-
OH
OH
OSO3
10c
(69%)
10f
(68%)
11e
O
O
O
BnO
BnO
BnO
HO
-O3SO
(42%)
HO
OBn
O
-O3SHN
O
-O3SHN
-O3SHN
O
NH2
NH2
O
BnO
NH2
BnO

R. Liu et al. / Carbohydrate Research 355 (2012) 19–27
23
36.8 mmol). The mixture was warmed to rt over 2 h and stirred for
another 10 h, then quenched with saturated aq NH4Cl (100 mL),
extracted with CH2Cl2 (3 ꢂ 200 mL), washed with brine (50 mL),
dried over Na2SO4 and concentrated, then purified by silica gel
chromatography (20% EtOAc in hexanes) to afford the 2,3-di-O-
benzyl ether as an amorphous white solid (7.7 g, 62% over three
steps).
2.20 mmol) and iPr2NEt (2.56 mL, 14.70 mmol), then SEM-Cl
(1.82 mL, 10.30 mmol). The mixture was stirred for 48 h at rt, then
extracted with CH2Cl2 (2 ꢂ 100 mL), washed with brine (40 mL),
dried over Na2SO4, then concentrated under reduced pressure.
Purification by silica gel chromatography (4.8% EtOAc in hexanes)
yielded 3-O-SEM ether 7 as an amorphous white solid (1.03 g,
91% over 2 steps). 1H NMR (400 MHz, CDCl3): d 7.81 (dd, 2H,
J = 1.2, 7.7 Hz), 7.71 (dd, 2H, J = 1.3, 9.1 Hz), 7.55 (d, 2H,
J = 8.1 Hz), 7.36 (m, 9H), 7.15 (dd, 2H, J = 3.6, 7.3 Hz), 7.05 (d, 2H,
J = 8.0 Hz), 4.98 (d, 1H, J = 6.4 Hz), 4.88 (d, 1H, J = 6.4 Hz), 4.79 (d,
1H, J = 10.8 Hz), 4.78 (d, 1H, J = 8.2 Hz), 4.69 (d, 1H, J = 10.6 Hz),
4.41 (d, 1H, J = 10.1 Hz), 4.01 (dd, 1H, J = 1.4, 11.5 Hz), 3.92 (dd,
1H, J = 3.1, 11.4 Hz), 3.70 (m, 5H), 3.30 (m, 2H), 2.33 (s, 3H), 1.11
(s, 9H), 0.97 (t, 2H, J = 8.3 Hz), 0.00 (s, 9H); 13C NMR (100 MHz,
CDCl3): d 138.50, 137.72, 135.83, 135.59, 134.05, 133.25, 132.73,
129.76, 128.42, 127.78, 127.71, 127.67, 127.37, 96.51, 86.33,
81.65, 79.91, 74.81, 66.40, 64.57, 62.23, 26.84, 21.15, 19.27,
18.08, –1.51; IR (NaCl): 2952, 2857, 2111, 1428, 1248, 1112,
The 4,6-anisylidene acetal (7.6 g, 13.8 mmol) was dissolved in
THF (274 mL) and treated at ꢀ78 °C with a 1 M solution of BH3
in THF (76.0 mmol) and a 1 M solution of Bu2BOTf in THF
(34.8 mL). The mixture was stirred at ꢀ78 °C for 10 h, then
quenched with Et3N (6.8 mL, 48.6 mmol) and MeOH (200 mL),
and warmed slowly to rt over 1 h. The product was extracted with
CH2Cl2 (3 ꢂ 200 mL), washed with brine (40 mL), dried over
Na2SO4, and concentrated, then purified by silica gel chromatogra-
phy (25% EtOAc in hexanes) to afford 6-O-PMB ether 4 as an amor-
phous white solid (7.5 g, 99%). 1H NMR (400 MHz, CDCl3): d 7.35
(m, 12H), 6.89 (d, 2H, J = 8.6 Hz), 4.99 (t, 2H, J = 11.5 Hz), 4.75 (d,
2H, J = 11.3 Hz), 4.52 (m, 2H), 4.46 (d, 1H, J = 7.3 Hz), 4.07 (ddd,
1H, J = 3.8, 5.4, 9.9 Hz), 3.81 (s, 3H), 3.60 (m, 9H), 2.60 (br, 1H);
13C NMR (100 MHz, CDCl3): d 159.23, 138.50, 138.31, 129.82,
129.35, 128.49, 128.34, 128.07, 127.90, 127.80, 127.67, 113.76,
103.62, 83.85, 81.63, 75.22, 74.77, 74.03, 73.25, 71.39, 69.77,
68.14, 55.21, 50.96; IR (NaCl): 3460, 2916, 2872, 2104, 1612,
1049 cmꢀ1; ½a 2D0
ꢃ
= –21.8 (c 1.35, CH2Cl2); HRESI-MS: m/z calcd for
C
43H55N3NaO5SSi2 [M+Na]+: 792.3299; found: 792.3313.
3.5. 20-(N-Carboxybenzyl)aminoethyl 4-O-[2-azido-4-O-benzyl-
6-O-(tert-butyldiphenylsilyl)-2-deoxy-3-O-[2-
(trimethylsilyl)ethoxymethyl]- -glucopyranosyl]-2,3-di-O-
a-
D
1513, 1248, 1064 cmꢀ1; ½a D20
ꢃ
= –13.2 (c 1.21, CH2Cl2); HRESI-MS:
benzyl-6-O-p-methoxybenzyl-b-D-glucopyranoside (1)
m/z calcd for C30H35N3NaO7 [M+Na]+: 572.2373; found: 572.2375.
Thioglycoside donor 7 (200 mg, 0.26 mmol) and glucoside
acceptor 5 (248 mg, 0.38 mmol) were dissolved in 1:2 (CH2Cl)2/
Et2O (6 mL) and treated with freshly activated 4A molecular sieves
(800 mg) for 1 h at rt under argon. The mixture was then cooled to
3.3. 20-(N-Carboxybenzyl)aminoethyl 2,3-di-O-benzyl-6-O-p-
methoxybenzyl-b-D-glucopyranoside (5)
A solution of 20-azidoethyl glucoside 4 (478 mg, 0.87 mmol) in
CH2Cl2 (20 mL) was treated with Bu3P (225 L, 0.91 mmol), and
ꢀ30 °C and treated with NIS (64 mg, 0.29 mmol) and TfOH (3.5
0.04 mmol). The mixture was stirred for 5 h at ꢀ30 °C, then
quenched with Et3N (22 L, 0.16 mmol), and filtered through Cel-
lL,
l
stirred at rt for 3 h, then treated with H2O (10 mL) and stirred at
rt for 1 day. The crude amine was concentrated and dried under re-
duced pressure with azeotropic distillation with toluene
(3 ꢂ 10 mL), to afford a yellowish oil. This was redispersed with
NaHCO3 (263 mg, 3.10 mmol) in 5:1 THF/H2O (10 mL), then treated
l
ite, which was rinsed with CH2Cl2 (3 ꢂ 70 mL). The filtrate was
washed with brine (40 mL), dried over Na2SO4, and concentrated,
then purified by silica gel chromatography (20% EtOAc in hexanes)
to yield orthogonally protected GlcN(a1?4)Glc disaccharide 1 as
at 0 °C with Cbz-Cl (135
l
L, 0.96 mmol). The reaction mixture was
an amorphous white solid (229 mg, 68%). 1H NMR (400 MHz,
CDCl3): d 7.69 (d, 2H, J = 6.7 Hz), 7.64 (d, 2H, J = 7.0 Hz), 7.30 (m,
26H), 7.00 (d, 2H, J = 8.2 Hz), 6.60 (d, 2H, J = 8.3 Hz), 5.70 (d, 1H,
J = 3.7 Hz), 5.60 (br, 1H), 5.04 (m, 5H), 4.89 (m, 5H), 4.72 (dd, 2H,
J = 5.7, 11.0 Hz), 4.42 (d, 1H, J = 7.9 Hz), 4.29 (m, 2H), 3.73 (m,
18H), 3.06 (dd, 1H, J = 3.8, 7.9 Hz), 1.07 (s, 9H), 1.01 (t, 2H,
J = 8.6 Hz), 0.04 (s, 9H); 13C NMR (100 MHz, CDCl3): d 158.92,
156.48, 138.43, 138.08, 137.99, 136.54, 135.84, 135.50, 133.55,
132.91, 129.60, 128.95, 128.42, 128.38, 128.11, 128.00, 127.92,
127.67, 127.53, 127.44, 113.53, 103.70, 97.66, 96.47, 84.71, 82.54,
78.11, 74.87, 74.72, 74.56, 74.00, 72.93, 72.18, 70.04, 68.88,
66.60, 66.19, 62.55, 61.86, 55.03, 41.51, 26.83, 19.28, 18.11,
ꢀ1.51; IR (NaCl): 3032, 2952, 2108, 1724, 1513, 1454, 1360,
warmed to rt over 2 h and stirred for another 10 h, then extracted
with CH2Cl2 (3 ꢂ 50 mL), washed with brine (20 mL), dried over
Na2SO4, and concentrated, and purified by silica gel chromatogra-
phy (33% EtOAc in hexanes) to afford Cbz-protected 20-aminoethyl
glucoside 5 as an amorphous white solid (526 mg, 92% over 2
steps). 1H NMR (400 MHz, CDCl3): d 7.36 (m, 15H), 7.24 (d, 2H,
J = 8.2 Hz), 6.88 (d, 2H, J = 8.2 Hz), 5.62 (br, 1H), 5.11 (s, 2H), 4.96
(d, 1H, J = 11.4 Hz), 4.90 (d, 1H, J = 11.1 Hz),4.78 (t, 2H,
J = 13.0 Hz), 4.99 (m, 2H), 4.41 (d, 1H, J = 7.1 Hz), 3.87 (m, 6H),
3.63 (m, 2H), 3.46 (m, 5H), 2.84 (s, 1H); 13C NMR (100 MHz, CDCl3):
d 159.23, 156.53, 138.54, 138.24, 136.56, 129.81, 129.36, 128.49,
128.47, 128.41, 128.11, 128.00, 127.91, 127.80, 127.74, 113.78,
103.83, 84.00, 81.66, 75.23, 74.83, 74.09, 73.16, 71.33, 69.87,
69.61, 66.64, 55.20, 41.42; IR (NaCl): 3360, 2872, 1711, 1513,
1248, 1112, 1040 cmꢀ1; ½a D20
ꢃ
= +55.0 (c 1.02, CH2Cl2); HRMALDI–
MS: m/z calcd for C73H90N4NaO14Si2 [M+Na]+: 1325.5889; found:
1325.5785.
1248, 1062 cmꢀ1; ½a D20
ꢃ
= –16.7 (c 0.65, CH2Cl2); HRESI-MS: m/z
calcd for C38H43NNaO9 [M+Na]+: 680.2836; found: 680.2835.
3.6. 20-(N-Carboxybenzyl)aminoethyl 4-O-[2-azido-4-O-benzyl-
6-O-(tert-butyldiphenylsilyl)-2-deoxy-3-O-[2-
3.4. Thiotolyl 2-azido-4-O-benzyl-6-O-(tert-butyldiphenylsilyl)-
2-deoxy-3-O-[2-(trimethylsilyl)ethoxymethyl]-b-
D
-
(trimethylsilyl)ethoxymethyl]-a-D-glucopyranosyl]-2,3-di-O-
glucopyranoside (7)
benzyl-b- -glucopyranoside (8)
D
3-O-Acetyl derivative 6 (1.0 g, 1.47 mmol) was prepared as pre-
6-O-PMB ether 1 (800 mg, 0.61 mmol) was dissolved in 90%
aqueous CH3CN (8 mL), treated at 0 °C with CAN (1 g, 1.84 mmol),
viously described,15 then dissolved in anhydrous MeOH (60 mL)
and treated at rt with 1 M NaOMe solution in MeOH (440
lL).
then stirred for 10 h. The mixture was quenched with Et3N (342 lL,
The mixture was stirred for 5 h at rt, neutralized with activated
Dowex 50X-W-H+ ion-exchange resin, filtered, concentrated, and
dried under reduced pressure. The crude C3 alcohol was redis-
solved in CH2Cl2 (40 mL) and treated at rt with TBAI (813 mg,
2.46 mmol), concentrated, and purified by silica gel chromatogra-
phy (20% EtOAc in hexanes) to afford C6 alcohol 8 as a colorless
oil (630 mg, 87%). 1H NMR (400 MHz, CDCl3): d 7.79 (dd, 4H,
J = 7.1, 8.4 Hz), 7.39 (m, 26H), 5.81 (d, 1H, J = 3.5 Hz), 5.55 (t, 1H,

24
R. Liu et al. / Carbohydrate Research 355 (2012) 19–27
J = 6.0 Hz), 5.16 (m, 4H), 4.99 (m, 3H), 4.92 (d, 1H, J = 10.8 Hz), 4.79
(d, 2H, J = 11.0 Hz), 4.52 (d, 1H, J = 7.7 Hz), 4.12 (t, 1H, J = 8.7 Hz),
3.13 (m, 13H), 3.47 (m, 5H), 3.20 (dd, 1H, J = 3.6, 10.4 Hz), 2.44 (t,
1H, J = 6.0 Hz), 1.18 (s, 9H), 1.10 (t, 3H, J = 8.4 Hz), 0.11 (s, 9H);
13C NMR (100 MHz, CDCl3): d 156.18, 138.30, 137.90, 137.62,
136.22, 135.68, 135.42, 133.23, 132.73, 129.51, 128.25, 127.98,
127.76, 127.60, 127.44, 127.32, 103.58, 97.82, 96.30, 84.43, 82.43,
78.08, 76.46, 74.79, 74.58, 72.42, 72.32, 69.36, 66.55, 66.06,
62.39, 62.18, 61.67, 41.05, 26.72, 19.13, 17.96, ꢀ1.60; IR (NaCl):
EtOAc (3 ꢂ 15 mL), washed with brine (10 mL), dried over Na2SO4,
and concentrated, then purified by silica gel chromatography (40%
EtOAc in hexanes) to yield the C30 alcohol as a white solid (70 mg,
93%). Op. 2: The intermediate azide (40 mg, 0.04 mmol) was dis-
solved in dry pyridine (0.58 mL), then treated with AcSH
(0.11 mL) and stirred at rt for 48 h. The mixture was concentrated
and purified by silica gel chromatography (3% MeOH in CHCl3) to
yield the C20 N-acetamide as an amorphous white solid (38 mg,
93%). Op. 3: The intermediate diol (38 mg, 0.04 mmol) was dis-
solved in dry DMF (1.2 mL), then treated with SO3ꢁMe3N (99 mg,
0.71 mmol) and stirred at 55 °C for 10 h. The reaction mixture
was concentrated and purified by silica gel chromatography (3%
MeOH in CHCl3 with 2% Et3N) to yield the 30,6-di-O-sulfate Et3NH
salt as a yellow solid (44 mg, 93%). Op. 4: The intermediate TBDPS
ether (45 mg, 0.04 mmol) was dissolved in DMF (0.4 mL), then
treated with a 1 M solution of TBAF in THF (0.4 mL) for 48 h and
concentrated to obtain a yellow oil. Op. 5: The crude disulfate ester
was dissolved in 50% aq MeOH and passed through an ion-ex-
change column, then concentrated, and purified by reverse-phase
HPLC to yield the disodium salt as an amorphous white solid
(20 mg, 55%). The overall yield of 30,6-di-O-sulfate 9b (disodium
salt) from 1 was 39% over 6 operations. 1H NMR (400 MHz,
CD3OD): d 7.45 (m, 2H), 7.23 (m, 18H), 5.49 (d, 1H, J = 3.4 Hz),
5.15 (d, 2H, J = 10.3 Hz), 4.99 (d, 2H, J = 7.4 Hz), 4.93 (d, 2H,
J = 11.2 Hz), 4.71 (m, 2H), 4.54 (m, 3H), 4.35 (dd, 1H, J = 2.3,
10.7 Hz), 4.23 (dd, 1H, J = 4.7, 10.9 Hz), 4.07 (dd, 1H, J = 3.5,
10.8 Hz), 3.94 (m, 1H), 3.95 (m, 3H), 3.76 (t, 1H, J = 8.6 Hz), 3.69
(dd, 3H, J = 4.1, 12.1 Hz), 3.59 (t, 1H, J = 9.4 Hz), 3.42 (t, 1H,
J = 8.3 Hz), 3.36 (dd, 2H, J = 5.3, 11.0 Hz), 1.76 (s, 3H); 13C NMR
(100 MHz, CD3OD): d 173.40, 158.86, 140.07, 139.84, 139.29,
138.24, 129.39, 129.28, 129.17, 129.07, 128.96, 128.87, 128.77,
128.64, 128.57, 128.39, 104.53, 98.67, 85.17, 83.54, 79.33, 75.86,
75.48, 75.26, 74.48, 74.38, 73.36, 72.00, 69.40, 68.09, 67.58,
2952, 2108, 1717, 1456, 1249, 1113, 1041 cmꢀ1; ½a D20
ꢃ
= +53.1 (c
1.02, CH2Cl2); ESI–MS: m/z calcd for C65H82N4NaO13Si2 [M+Na]+:
1205.5; found: 1205.4.
3.7. 20-(N-Carboxybenzyl)aminoethyl 4-O-[2-acetamido-4-O-
benzyl-2-deoxy-6-O-sulfonato-a-D-glucopyranosyl]-2,3-di-O-
benzyl-6-O-sulfonato-b- -glucopyranoside (9a)
D
Op. 1: Orthogonally protected disaccharide
8
(70 mg,
0.06 mmol) was dissolved in THF (0.85 mL), then treated with a
1 M solution of TBAF in THF (0.35 mL) and stirred at rt for 6 h.
The mixture was concentrated then purified by silica gel chroma-
tography (40% EtOAc in hexanes) to yield the C60 alcohol as a white
solid (49 mg, 87%). Op. 2: The intermediate azide (40 mg,
0.04 mmol) was dissolved in dry pyridine (0.6 mL), then treated
with AcSH (121 lL, 1.69 mmol) and stirred at rt for 48 h. The mix-
ture was concentrated and purified by silica gel chromatography
(3% MeOH in CHCl3) to yield the C20 N-acetamide as an amorphous
white solid (38 mg, 95%). Op. 3: The intermediate diol (34 mg,
0.04 mmol) was dissolved in dry DMF (1.1 mL), then treated with
SO3ꢁMe3N (98 mg, 0.70 mmol) and stirred at 55 °C for 10 h. The
reaction mixture was concentrated and purified by silica gel chro-
matography (5% MeOH in CHCl3 with 2% Et3N) to yield the 60,6-di-
O-sulfate Et3NH salt as a yellow solid (44 mg, 93%). Op. 4: The
intermediate SEM ether (20 mg, 0.02 mmol) was dissolved in
Et2O (1 mL), then treated with a solution of MgBr2ꢁEt2O (46 mg,
0.18 mmol) in CH3NO2 (0.9 mL) and Et2O (0.8 mL) and stirred at
rt for 48 h. The reaction mixture was quenched with saturated
NaHCO3 (2 mL), extracted with CH2Cl2 (3 ꢂ 2 mL), washed with
brine (2 mL), dried over Na2SO4, and concentrated. Op. 5: The crude
disulfate ester was dissolved in 50% aq MeOH and passed through
an ion-exchange column, then concentrated and purified by re-
verse-phase HPLC to yield the disodium salt as an amorphous
white solid (14 mg, 79%). The overall yield of 60,6-di-O-sulfate 9a
(disodium salt) from 1 was 52% over 6 operations. 1H NMR
(400 MHz, CD3OD): d 7.44 (d, 3H, J = 7.4 Hz), 7.27 (m, 17H), 5.44
(d, 1H, J = 3.9 Hz), 4.98 (d, 1H, J = 5.5 Hz), 4.91 (s, 2H), 4.81 (d,
2H, J = 2.1 Hz), 4.56 (t, 2H, J = 10.4 Hz), 4.50 (d, 1H, J = 7.7 Hz),
4.34 (m, 3H), 4.13 (dd, 1H, J = 5.6, 11.2 Hz), 4.04 (dd, 1H, J = 3.7,
10.7 Hz), 3.92 (m, 2H), 3.70 (m, 6H), 3.56 (t, 1H, J = 9.5 Hz), 3.43
(m, 1H), 1.80 (s, 3H); 13C NMR (100 MHz, CD3OD) d 173.82,
158.86, 139.83, 138.25, 129.87, 129.54, 129.39, 129.21, 129.13,
129.08, 128.88, 128.78, 128.66, 128.50, 128.34, 104.46, 99.22,
85.02, 83.56, 79.49, 77.76, 75.82, 75.72, 75.40, 75.27, 74.49,
67.39, 55.11, 41.95, 22.64; ½a D20
ꢃ
= +78.9 (c 1.0, MeOH); HRESI-MS:
m/z calcd for C45H52N2Na3O19S2 [M+3Na]+: 1057.2299; found:
1057.2288.
3.9. 20-(N-Carboxybenzyl)aminoethyl 4-O-[2-amino-4-O-benzyl-
2-deoxy-2-N-sulfonato-a-D-glucopyranosyl]-2,3-di-O-benzyl-6-
O-sulfonato-b- -glucopyranoside (9c)
D
Op. 1: Orthogonally protected disaccharide
8
(40 mg,
0.03 mmol) was dissolved in dry DMF (1.13 mL), then treated with
SO3ꢁMe3N (98 mg, 0.34 mmol) at 55 °C for 10 h. The mixture was
concentrated and purified by silica gel chromatography (2% MeOH
in CHCl3 with 2% Et3N) to yield the 6-O-sulfate Et3NH salt as a yel-
low solid (40 mg, 90%). Op. 2: The intermediate sulfoform (40 mg,
0.03 mmol) was dissolved in Et2O (2 mL), then treated with
MgBr2ꢁEt2O (80 mg, 0.30 mol) in CH3NO2 (1.5 mL) and Et2O
(1 mL) and stirred at rt for 24 h. The mixture was quenched with
saturated NaHCO3 (10 mL), extracted with CH2Cl2 (3 ꢂ 15 mL),
washed with brine (10 mL), dried over Na2SO4, and concentrated,
then purified by silica gel chromatography (5% MeOH in CHCl3
with 2% Et3N) to yield the C30 alcohol as a yellow solid (40 mg,
90%). Op. 3: The intermediate TBDPS ether (30 mg, 0.02 mmol)
was dissolved in DMF (1 mL), then treated with a 1 M solution of
TBAF in THF (0.19 mL) and stirred at rt for 24 h. The mixture
was concentrated and purified by silica gel chromatography (9%
MeOH in CHCl3 with 2% Et3N) to yield the C60 alcohol as a yellow
solid (19 mg, 78%). Op. 4: The intermediate azide (28 mg,
0.03 mmol) was dissolved in MeOH (1 mL), then treated with
73.99, 69.37, 67.86, 67.39, 62.01, 54.77, 41.97, 22.97; ½a D20
ꢃ
= +73.6
C45H52N2Na3O19S2
(c 1.0, MeOH); HRESI-MS: m/z calcd for
[M+3Na]+: 1057.2299; found: 1057.2282.
3.8. 20-(N-Carboxybenzyl)aminoethyl 4-O-[2-acetamido-4-O-
benzyl-2-deoxy-3-O-sulfonato- -glucopyranosyl]-2,3-di-O-
benzyl-6-O-sulfonato-b- -glucopyranoside (9b)
a-D
D
Op. 1: Orthogonally protected disaccharide
8
(85 mg,
1,3-propanedithiol (130 lL, 0.93 mmol) and Et3N (94 lL,
0.07 mmol) was dissolved in Et2O (3 mL), then treated with a solu-
tion of MgBr2ꢁEt2O (185 mg, 0.72 mmol) in CH3NO2 (3 mL) and
Et2O (3 mL) and stirred at rt for 48 h. The reaction mixture was
quenched with saturated aq NaHCO3 (10 mL), extracted with
0.93 mmol) and stirred at rt for 24 h. The mixture was concen-
trated and purified by silica gel chromatography (10% MeOH in
CHCl3 with 2% Et3N) to yield the C20 amine as a yellow solid
(25 mg, 93%). Op. 5: The amine (48 mg, 0.56 mmol) was dissolved

R. Liu et al. / Carbohydrate Research 355 (2012) 19–27
25
in DMF (1 mL) and mixed with aq NaOH adjusted to pH 9.5 (5 mL),
3.11. 20-(N-Carboxybenzyl)aminoethyl 4-O-[2-amino-4-O-
benzyl-2-deoxy-2,6-di-N,O-sulfonato- -glucopyranosyl]-2,3-
di-O-benzyl-6-O-sulfonato-b- -glucopyranoside (9e)
then treated with SO3ꢁPy (162 mg, 5.60 mmol), stirred at rt for 8 h,
and concentrated. Op. 6: The crude di-N,O-sulfate ester was
dissolved in 50% aq MeOH and passed through an ion-exchange
column, then concentrated, and purified by reverse-phase HPLC
to yield the disodium salt as an amorphous white solid (33 mg,
63%). The overall yield of 20,6-di-N,O-sulfate 9c (disodium salt)
from 1 was 36% over 7 operations. 1H NMR (400 MHz, CD3OD): d
7.07–7.44 (m, 20H), 5.52 (d, 1H, J = 3.5 Hz), 4.99 (d, 1H,
J = 3.9 Hz), 4.95 (d, 1H, J = 4.6 Hz), 4.82–4.93 (m, 2H), 4.64 (d, 1H,
J = 11.0 Hz), 4.56 (d, 1H, J = 11.2 Hz), 4.49 (d, 1H, J = 7.5 Hz), 4.38
(dd, 1H, J = 2.6, 10.9 Hz), 4.16 (dd, 1H, J = 5.3, 10.7 Hz), 3.81–3.98
(m, 3H), 3.58–3.80 (m, 7H), 3.42 (m, 2H), 3.32–3.37 (m, 3H); 13C
a-D
D
Op. 1: Orthogonally protected disaccharide 8 was treated with
TBAF in THF to yield the C60 alcohol, as described in the synthesis
of 9a. Op. 2: The intermediate azide (80 mg, 0.09 mmol) was dis-
solved in MeOH (3 mL), treated with 1,3-propanedithiol (84
lL,
0.85 mmol) and Et3N (118 L, 0.85 mmol), then stirred at rt for
l
40 h. The mixture was concentrated and purified by silica gel chro-
matography (3.25% MeOH in CH2Cl2) to afford the C2 amine as an
amorphous white solid (68 mg, 87%). Op. 3: The intermediate ami-
no diol (60 mg, 0.07 mmol) was dissolved in 5:1 Py/Et3N (3 mL)
and treated with SO3ꢁPy (281 mg, 1.76 mmol), then heated to
70 °C under microwave conditions for 1 h. The mixture was con-
centrated and purified by silica gel chromatography (9% MeOH in
CH2Cl2 with 2% Et3N) to yield the 20,60,6-tri-N,O,O-sulfate Et3NH
salt as a light yellow oil. Op. 4: The intermediate SEM ether was
dispersed in Et2O (4 mL), treated with a solution of MgBr2ꢁEt2O
(169 mg, 0.65 mmol) in CH3NO2 (3 mL, 55.80 mmol) in Et2O
(2 mL), then stirred at rt for 40 h. The mixture was concentrated
and used directly in the next operation. Op. 5: The crude trisulfate
ester was dissolved in 50% aq MeOH and passed through an ion-ex-
change column, then purified by reverse-phase HPLC to yield the
trisodium salt as an amorphous white solid (46 mg, 64% over 2
steps). The overall yield of 20,60,6-tri-N,O,O-sulfate 9e (trisodium
salt) from 1 was 42% over 6 operations. 1H NMR (500 MHz,
CD3OD): d 7.45 (d, 2H, J = 7.1 Hz), 7.36 (d, 2H, J = 7.2 Hz), 7.15–
7.32 (m, 16H), 5.57 (d, 1H, J = 3.1 Hz), 4.91–5.03 (m, 4H), 4.83 (d,
1H, J = 11.2 Hz), 4.77 (d, 1H, J = 10.5 Hz), 4.58 (d, 1H, J = 13.2 Hz),
4.56 (d, 1H, J = 11.2 Hz), 4.50 (d, 1H, J = 7.3 Hz), 4.27–4.40 (m,
3H), 4.20 (dd, 1H, J = 5.0, 10.8 Hz), 3.84–3.98 (m, 3H), 3.73–3.83
(m, 2H), 3.67 (m, 2H), 3.57 (m, 2H), 3.44 (t, 1H, J = 7.7 Hz), 3.33–
3.40 (m, 3H), 1.94 (s, 1H); 13C NMR (125 MHz, CD3OD): d 158.99,
140.29, 139.99, 139.82, 138.36, 129.62, 129.58, 129.38, 129.29,
129.24, 129.06, 128.94, 128.67, 128.57, 128.50, 104.64, 99.27,
83.78, 83.43, 79.24, 75.76, 75.35, 75.29, 75.00, 74.60, 71.47,
NMR (100 MHz, CD3OD)
d 158.84, 140.23, 139.87, 139.55,
138.25, 129.64, 129.53, 129.40, 129.25, 129.14, 129.03, 128.88,
128.78, 128.50, 128.41, 104.50, 99.48, 83.41, 79.42, 75.74, 75.19,
75.15, 75.04, 74.94, 74.43, 73.28, 69.41, 68.03, 67.40, 62.26,
60.15, 41.97;
½
a 2D0
= +53.2 (c 0.2, MeOH); HRESI-MS:
ꢃ
m/z calcd for C43H50N2Na3O18S2 [M+3Na]+: 1015.2193; found:
1057.2209.
3.10. 20-(N-Carboxybenzyl)aminoethyl 4-O-[2-acetamido-4-O-
benzyl-2-deoxy-3,6-di-O-sulfonato-a-D-glucopyranosyl]-2,3-di-
O-benzyl-6-O-sulfonato-b- -glucopyranoside (9d)
D
Op. 1: Orthogonally protected disaccharide 8 was treated with
TBAF in THF to yield the C60 alcohol, as described in the synthesis
of 9a. Op. 2: The intermediate SEM ether (160 mg, 0.17 mmol) was
dissolved in Et2O (7 mL), then treated with
a
solution of
MgBr2ꢁEt2O (437 mg, 1.69 mmol) and CH3NO2 (182
lL, 3.38 mmol)
in Et2O (5 mL) and stirred at rt for 10 h. The mixture was quenched
with saturated aq NaHCO3 (70 mL), extracted with EtOAc
(3 ꢂ 100 mL), washed with brine (20 mL), dried over Na2SO4, and
concentrated, then purified by silica gel chromatography (66%
EtOAc in hexanes) to yield the C30 alcohol as an amorphous white
solid (134 mg, 97%). Op. 3: The intermediate azide (67 mg,
0.08 mmol) was dissolved in pyridine (2 mL), then treated with
AcSH (234
lL, 3.29 mmol) and stirred at rt for 40 h. The mixture
69.59, 68.15, 67.72, 67.55, 60.06, 47.99, 42.10; ½a D20
ꢃ
= +33.6 (c
was concentrated and purified by silica gel chromatography
(4.75% MeOH in CH2Cl2) to yield the C20 N-acetamide as an amor-
phous white solid (60 mg, 88%). Op. 4: The intermediate amino
alcohol (44 mg, 0.05 mmol) was dissolved in 5:1 Py/Et3N (3 mL)
and treated with SO3ꢁPy (228 mg, 1.43 mmol), then heated to
70 °C under microwave conditions for 1 h. The mixture was con-
centrated and purified by silica gel chromatography (9% MeOH
in CH2Cl2 with 2% Et3N) to yield the 30,60,6-tri-O-sulfate Et3NH salt
as a yellow solid. Op. 5: The crude trisulfate ester was dissolved in
50% aq MeOH and passed through an ion-exchange column, then
concentrated and purified by reverse-phase HPLC to yield the tri-
sodium salt as an amorphous white solid (45 mg, 74%). The overall
yield of 30,60,6-tri-O-sulfate 9d (trisodium salt) from 1 was 48%
over 6 operations. 1H NMR (500 MHz, CD3OD): d 7.56 (d, 2H,
J = 7.2 Hz), 7.16–7.34 (m, 18H), 5.54 (d, 1H, J = 2.0 Hz), 5.12 (d,
1H, J = 9.6 Hz), 4.99 (m, 2H), 4.92 (d, 1H, J = 11.2 Hz), 4.68 (m,
3H), 4.54 (d, 1H, J = 12.0 Hz), 4.52 (d, 1H, J = 8.3 Hz), 4.40 (d, 1H,
J = 10.0 Hz), 4.35 (m, 2H), 4.22 (dd, 1H, J = 4.0, 10.6 Hz), 4.11 (dd,
1H, J = 2.4, 8.4 Hz), 3.91–4.02 (m, 2H), 3.85 (t, 1H, J = 8.8 Hz),
3.78 (t, 1H, J = 8.4 Hz), 3.69 (m, 7H), 3.56 (t, 1H, J = 4.4 Hz), 3.42
(t, 1H, J = 7.9 Hz), 3.37 (br, 1H), 1.94 (s, 3H); 13C NMR (125 MHz,
0.93, MeOH); ESI–MS: m/z calcd for C43H49N2Na4O21S3 [M+4Na]+:
1117.2; found: 1117.0.
3.12. 20-(N-Carboxybenzyl)aminoethyl 4-O-[2-amino-4-O-
benzyl-2-deoxy-2,3-di-N,O-sulfonato-
di-O-benzyl-6-O-sulfonato-b- -glucopyranoside (9f)
a-D-glucopyranosyl]-2,3-
D
Op. 1: Orthogonally protected disaccharide
0.13 mmol) was dissolved in Et2O (20 mL), then treated with a
solution of MgBr2ꢁEt2O (441 mg, 1.34 mmol) and CH3NO2 (143 L,
8
(158 mg,
l
2.68 mmol) in Et2O (6 mL) and stirred for 10 h. The mixture was
quenched with saturated aq NaHCO3 (70 mL), extracted with
EtOAc (3 ꢂ 100 mL), washed with brine (20 mL), dried over
Na2SO4, and concentrated, then purified by silica gel chromatogra-
phy (66% EtOAc in hexanes) to afford an amorphous white solid
(136 mg, 96%). Op. 2: The intermediate azide (136 mg, 0.13 mmol)
was dissolved in MeOH (3 mL), then treated with 1,3-propanedi-
thiol (133 lL, 1.30 mmol) and Et3N (186 lL, 1.30 mmol) and stirred
at rt for 40 h. The mixture was concentrated and purified by silica
gel chromatography (4.75% MeOH in CH2Cl2) to afford the C20
amine as an amorphous white solid (101 mg, 77%). Op. 3: A solu-
tion of amino diol (93 mg, 0.09 mmol) was dissolved in 5:1 Py/
Et3N (3 mL) and treated with SO3ꢁPy (385 mg, 2.42 mmol), then
heated to 70 °C under microwave conditions for 1 h. The mixture
was concentrated and purified by silica gel chromatography (9%
MeOH in CH2Cl2 with 2% Et3N) to yield 20,30,6-tri-N,O,O-sulfate
Et3NH salt as a light yellow oil. Op. 4: The intermediate TBDPS
ether was redissolved in DMF (2 mL), then treated with 1 M TBAF
CD3OD):
d 173.98, 159.04, 139.99, 139.94, 139.77, 138.40,
130.70, 129.57, 129.40, 129.30, 129.19, 129.07, 128.95, 128.74,
128.51, 104.63, 99.03, 85.21, 83.71, 79.60, 77.58, 76.19, 75.59,
75.28, 75.24, 74.45, 73.59, 72.25, 69.57, 68.05, 67.56, 67.49,
62.32, 55.26, 54.68, 42.12, 23.18; ½a D20
ꢃ
= +25.0 (c 0.79, MeOH);
ESI–MS: m/z calcd for C45H51N2Na4O22S3 [M+4Na]+: 1159.2; found:
1159.1.

26
R. Liu et al. / Carbohydrate Research 355 (2012) 19–27
solution in THF (0.9 mL) and stirred at rt for 40 h. The mixture was
concentrated and used directly in the next operation. Op. 5: The
crude trisulfate ester was dissolved in 50% aq MeOH and passed
through an ion-exchange column, then purified by reverse-phase
HPLC to yield the trisodium salt as an amorphous white solid
(61 mg, 62%). The overall yield of 20,30,6-tri-N,O,O-sulfate 9f (triso-
dium salt) from 1 was 40% over 6 operations. 1H NMR (400 MHz,
CD3OD): d 7.46 (dd, 2H, J = 1.4, 8.3 Hz), 7.11–7.42 (m, 18H), 5.75
(d, 1H, J = 3.4 Hz), 5.14 (d, 1H, J = 10.8 Hz), 5.10 (d, 1H,
J = 11.1 Hz), 4.99 (m, 2H), 4.82 (d, 1H, J = 11.2 Hz), 4.65 (dd, 1H,
J = 8.9, 10.5 Hz), 4.58 (d, 1H, J = 10.6 Hz), 4.54 (d, 1H, J = 11.2 Hz),
4.52 (d, 1H, J = 7.3 Hz), 4.37 (dd, 1H, J = 2.0, 10.8 Hz), 4.24 (dd,
1H, J = 4.8, 10.7 Hz), 3.53–4.05 (m, 10H), 3.49 (dd, 1H, J = 3.4,
10.6 Hz), 3.33–3.44 (m, 3H), 1.94 (s, 2H); 13C NMR (100 MHz,
CD3OD): d 158.86, 140.36, 139.98, 139.74, 138.26, 129.93, 129.40,
129.33, 129.17, 129.07, 128.97, 128.90, 128.75, 128.59, 128.40,
128.06, 104.35, 98.36, 84.57, 83.58, 79.47, 78.25, 75.64, 75.19,
74.95, 74.51, 74.15, 73.66, 69.30, 68.13, 67.38, 62.18, 58.86,
reverse-phase HPLC to yield 60,6-di-O-sulfate 2a (disodium salt)
as an amorphous white solid (10.5 mg, 87%). 1H NMR (400 MHz,
D2O): d 5.38 (d, 1H, J = 3.6 Hz) 4.42 (d, 1H, J = 8.0 Hz), 4.26 (m,
4H), 4.15 (dd, 1H, J = 5.1, 11.4 Hz), 3.88 (d, 2H, J = 3.6 Hz), 3.84
(m, 3H), 3.73 (m, 1H) 3.68 (m, 1H) 3.63 (t, 2H, J = 8.8 Hz), 3.54 (t,
1H, J = 9.5 Hz), 3.24 (m, 1H), 2.98 (t, 1H, J = 5.2 Hz), 1.96 (s, 3H);
13C NMR (100 MHz, D2O): d 174.98, 102.66, 98.02, 76.91, 75.37,
73.93, 73.03, 71.31, 71.17, 69.71, 69.47, 67.74, 67.16, 54.20,
40.42, 22.53; ½a D20
ꢃ
= +56.0 (c 0.5, H2O); HRESI-MS: m/z calcd for
C
16H28N2NaO17S2 [M+Na]ꢀ: 607.0727; found: 607.0733.
3.15. 20-Aminoethyl 4-O-[2-acetamido-2-deoxy-3-O-sulfonato-
-glucopyranosyl]-6-O-sulfonato-b- -glucopyranoside (2b)
a-D
D
Partially protected sulfoform 9b (20 mg, 0.02 mmol) was dis-
solved in 50% aq MeOH (1 mL), then treated with 10% Pd(OH)2
on charcoal (8 mg) and stirred under positive H2 pressure at rt
for 20 h. The product was filtered, concentrated, and purified by re-
verse-phase HPLC to yield 30,6-di-O-sulfate 2b (disodium salt) as
an amorphous white solid (10 mg, 83%). 1H NMR (400 MHz,
D2O): d 5.30 (d, 1H, J = 3.7 Hz), 4.44 (m, 1H), 4.36 (m, 1H), 4.29
(dd, 1H, J = 2.1, 11.3 Hz), 4.18 (dd, 1H, J = 4.5, 11.2 Hz), 4.01 (dd,
1H, J = 3.6, 10.8 Hz), 3.90 (dd, 1H, J = 5.4, 11.0 Hz), 3.78 (dd, 2H,
J = 2.9, 7.4 Hz), 3.70 (m, 3H), 3.62 (m, 3H), 3.22 (m, 1H) 2.93 (t,
1H, J = 5.3 Hz), 1.93 (s, 3H); 13C NMR (100 MHz, D2O): d 174.91,
102.69, 99.28, 80.18, 76.80, 76.58, 73.93, 73.23, 73.08, 70.09,
41.97;
C
½
a 2D0
= +54.4 (c 0.62, MeOH); ESI–MS: m/z calcd for
ꢃ
43H49N2Na4O21S3 [M+4Na]+: 1117.2; found: 1117.0.
3.13. 20-(N-Carboxybenzyl)aminoethyl 4-O-[2-amino-4-O-
benzyl-2-deoxy-2,3,6-tri-N,O,O-sulfonato- -glucopyranosyl]-
2,3-di-O-benzyl-6-O-sulfonato-b- -glucopyranoside (9g)
a-D
D
Op. 1: Orthogonally protected disaccharide 8 was treated with
TBAF in THF to yield the C6’ alcohol, as described in the synthesis
of 9a. Op. 2: The intermediate SEM ether was treated with
MgBr2ꢁEt2O and CH3NO2 in Et2O to yield the C30 alcohol, as de-
scribed in the synthesis of 9d. Op. 3: The intermediate azide
(68 mg, 0.08 mmol) was dissolved in MeOH (3 mL), then treated
68.64, 67.52, 60.64, 53.10, 40.49, 22.73; ½a D20
ꢃ
= +31.1 (c 0.45,
H2O); HRESI-MS: m/z calcd for C16H28N2NaO17S2 [M+Na]ꢀ:
607.0727; found: 607.0722.
3.16. 20-Aminoethyl 4-O-[2-amino-2-deoxy-2-N-sulfonato-
glucopyranosyl]-6-O-sulfonato-b- -glucopyranoside (2c)
a-D-
with 1,3-propanedithiol (101
lL, 1.67 mmol) and Et3N (232
lL,
D
1.67 mmol) and stirred at rt for 20 h. The mixture was concen-
trated and purified by silica gel chromatography (4.75% MeOH in
CH2Cl2) to yield the C2’ amine as an amorphous white solid
(62 mg, 94%). Op. 4: A solution of amino triol (76 mg, 0.10 mmol)
was dissolved in 5:1 Py:Et3N (3 mL) and treated with SO3ꢁPy
(552 mg, 3.47 mmol), then heated to 70 °C under microwave con-
ditions for 1 h. The mixture was concentrated and purified by silica
gel chromatography (9% MeOH in CH2Cl2 with 2% Et3N) to yield
20,30,60,6-tetra-N,O,O,O-sulfate Et3NH salt as a light yellow oil. Op.
4: The tetrasulfate ester was dissolved in 50% aq MeOH and passed
through an ion-exchange column, then purified by reverse-phase
HPLC to yield the tetrasodium salt as an amorphous white solid
(90 mg, 78% over 2 steps). The overall yield of 20,30,60,6-tetra-
N,O,O,O-sulfate 9 g (tetrasodium salt) from 1 was 54% over 6
operations.1H NMR (400 MHz, CD3OD): 7.57 (d, 2H, J = 7.3 Hz),
7.06–7.49 (m, 18H), 5.51 (d, 1H, J = 2.6 Hz), 5.13 (d, 1H,
J = 9.5 Hz), 5.06 (d, 1H, J = 11.2 Hz), 4.98 (m, 2H), 4.89 (d, 1H,
J = 11.7 Hz), 4.70 (d, 1H, J = 9.5 Hz), 4.63 (t, 1H, J = 9.8 Hz), 4.46–
4.60 (m, 3H), 4.23–4.42 (m, 4H), 3.84–4.06 (m, 4H), 3.49–3.84
(m, 6H), 3.41 (t, 1H, J = 8.0 Hz), 3.33 (br, 1H); 13C NMR (125 MHz,
CD3OD): 157.34, 138.84, 138.38, 137.93, 136.68, 129.12, 127.94,
127.73, 127.68, 127.61, 127.55, 127.53, 127.45, 127.31, 127.24,
127.00, 126.58, 102.88, 96.62, 82.75, 82.05, 78.36, 76.26, 74.55,
73.84, 73.07, 72.37, 71.79, 70.22, 67.93, 66.74, 65.95, 57.07,
Partially protected sulfoform 9c (20 mg, 0.02 mmol) was dis-
solved in 50% aq MeOH (1 mL), then treated with 10% Pd(OH)2
on charcoal (8 mg) and stirred under positive H2 pressure at rt
for 20 h. The product was filtered, concentrated, and purified by re-
verse-phase HPLC to yield 20,6-di-N,O-sulfate 2c (disodium salt) as
an amorphous white solid (10 mg, 84%). 1H NMR (400 MHz, D2O): d
5.50 (d, 1H, J = 3.5 Hz), 4.44 (d, 1H, J = 8.1 Hz), 4.28 (dd, 1H, J = 1.9,
11.2 Hz), 4.13 (dd, 1H, J = 4.9, 11.5 Hz), 3.97 (m, 1H), 3.89 (m, 1H),
3.78 (dd, 1H, J = 2.4, 12.6 Hz), 3.71 (m, 3H), 3.66 (m, 2H), 3.52 (t,
1H, J = 9.5 Hz), 3.43 (t, 1H, J = 9.5 Hz), 3.27 (m, 1H), 3.14 (m, 3H);
13C NMR (100 MHz, D2O): d 102.57, 98.98, 76.54, 76.15, 73.35,
73.10, 73.04, 71.74, 70.10, 67.60, 67.11, 60.80, 58.72, 40.13;
½
a 2D0
ꢃ
= +26.2 (c 0.8, H2O); HRESI-MS: m/z calcd for
C
14H26N2NaO16S2 [M+Na]ꢀ: 565.0622; found: 565.0613.
3.17. 20-Aminoethyl 4-O-[2-acetamido-2-deoxy-3,6-di-O-
sulfonato- -glucopyranosyl]-6-O-sulfonato-b-
glucopyranoside (2d)
a-
D
D-
Partially protected sulfoform 9d (20.3 mg, 0.02 mmol) was dis-
solved in 50% aq MeOH (3 mL), then treated with 10% Pd(OH)2 on
charcoal (20 mg) and stirred under positive H2 pressure at rt for
20 h. The product was filtered, concentrated, and purified by re-
verse-phase HPLC to yield 30,60,6-tri-O-sulfate 2d (trisodium salt)
as an amorphous white solid (12.7 mg, 97%). 1H NMR (400 MHz,
D2O): d 5.32 (d, 1H, J = 3.6 Hz), 4.43 (d, 1H, J = 8.0 Hz), 4.36 (t, 1H,
J = 10.4 Hz), 4.15–4.30 (m, 5H), 4.02 (dd, 1H, J = 3.6, 10.7 Hz),
3.86–3.98 (m, 3H), 3.70–3.77 (m, 1H), 3.59–3.69 (m, 3H), 3.25 (t,
1H, J = 8.4 Hz), 3.08–3.21 (m, 2H), 1.92 (s, 3H); 13C NMR
(125 MHz, D2O): d 174.21, 101.86, 98.17, 79.24, 75.83, 75.58,
73.11, 72.36, 70.69, 67.59, 66.84, 66.42, 66.12, 52.27, 39.42,
40.42;
C
½
a 2D0
= +71.9 (c 0.98, MeOH); ESI–MS: m/z calcd for
ꢃ
43H48N2Na5O24S4 [M+5Na]+: 1219.1; found: 1219.1.
3.14. 20-Aminoethyl 4-O-[2-acetamido-2-deoxy-6-O-sulfonato-
-glucopyranosyl]-6-O-sulfonato-b- -glucopyranoside (2a)
a-
D
D
Partially protected sulfoform 9a (20 mg, 0.02 mmol) was dis-
solved in 50% aq MeOH (1 mL), then treated with 10% Pd(OH)2
on charcoal (8 mg) and stirred under positive H2 pressure at rt
for 20 h. The product was filtered, concentrated, and purified by
22.04;
C
½
a 2D0
= +39.2 (c 0.85, H2O); HRESI-MS: m/z calcd for
ꢃ
16H27N2Na4O20S3 [M+4Na]+: 754.9910; found: 754.9918.

R. Liu et al. / Carbohydrate Research 355 (2012) 19–27
27
3.18. 20-Aminoethyl 4-O-[2-acetamido-2-deoxy-2,6-di-O-
sulfonato- -glucopyranosyl]-6-O-sulfonato-b-
glucopyranoside (2e)
78.10, 76.52, 75.79, 72.49, 72.29, 70.56, 67.73, 66.90, 66.32,
a
-D
D-
55.87, 39.50; ½a D20
ꢃ
= +34.6 (c 1.0, H2O); HRESI-MS: m/z calcd for
C
14H24N2Na5O22S4 [M+5Na]+: 814.9192; found: 814.9194.
Partially protected sulfoform 9e (24.4 mg, 0.02 mmol) was dis-
solved in 50% aq MeOH (3 mL), then treated with 10% Pd(OH)2 on
charcoal (24 mg) and stirred under positive H2 pressure at rt for
20 h. The product was filtered, concentrated, and purified by re-
verse-phase HPLC to yield 20,60,6-tri-N,O,O-sulfate 2e (trisodium
salt) as an amorphous white solid (15.1 mg, 98%). 1H NMR
(400 MHz, D2O): d 5.65 (d, 1H, J = 3.6 Hz), 4.56 (t, 1H, J = 7.7 Hz),
4.31–4.38 (m, 3H), 4.25 (dd, 1H, J = 4.9, 11.4 Hz), 4.00–4.19 (m,
2H), 3.80–3.90 (m, 3H), 3.74 (t, 1H, J = 9.1 Hz), 3.61 (m, 2H),
3.34–3.48 (m, 2H), 3.30 (m, 2H), 2.91 (s, 2H), 2.76 (s, 2H); 13C
NMR (125 MHz, D2O): d 101.90, 97.94, 75.87, 75.06, 72.62, 72.40,
Acknowledgments
This work was supported by Grants from the National Institutes
of Health (GM-06982) and the Department of Defense (W911SR-
08-C-0001). The authors also gratefully acknowledge NMR and
MS support from the Purdue University Center for Cancer Research.
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4.28 (m, 2H), 4.13 (dd, 1H, J = 4.8, 11.3 Hz), 3.90–4.07 (m, 2H),
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Partially protected sulfoform 9g (47.9 mg, 0.04 mmol) was dis-
solved in 50% aq MeOH (3 mL), then treated with 10% Pd(OH)2 on
charcoal (24 mg) and stirred under positive H2 pressure at rt for
20 h. The product was filtered, concentrated, and purified by re-
verse-phase HPLC to yield 20,30,60,6-tetra-N,O,O,O-sulfate 2g (tetra-
sodium salt) as an amorphous white solid (31.4 mg, 99%). 1H NMR
(400 MHz, D2O): d 5.55 (d, 1H, J = 3.0 Hz), 4.59 (d, 1H, J = 8.0 Hz),
4.25–4.44 (m, 5H), 3.97–4.18 (m, 3H), 3.68–3.96 (m, 5H), 3.16–
3.50 (m, 4H); 13C NMR (125 MHz, D2O): d 101.94, 98.75, 81.70,