The synthesis of a multivalent heterobifunctional ligand for specific interaction with Shiga toxin 2 produced by E. coli O157:H7

Article abstract of DOI:10.1016/j.carres.2013.05.010

Hemolytic uremic syndrome is a potentially fatal complication of food poisoning caused by Escherichia coli O157:H7, especially those strains that produce the Stx2 Shiga toxin. Multivalent inhibitors based on the P^k trisaccharide are most effective against Stx1 the less dangerous of the two Shiga toxins. Inhibitors containing a terminal 2-acetamido-2-deoxy-α-D- galactopyranosyl residue in place of the terminal α-D-galactopyranosyl residue of P^k trisaccharide have been shown to exhibit preferential binding to Stx2. A multivalent heterobifunctional P^k analog containing 2-acetamido-2-deoxy-α-D-galactopyranose has been synthesized in a format that facilitates the ablation of toxin activity via supramolecular complex formation between Stx and the endogenous protein, Human serum amyloid P component (HuSAP).

Full text of DOI:10.1016/j.carres.2013.05.010

Carbohydrate Research 378 (2013) 4–14
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
The synthesis of a multivalent heterobifunctional ligand for specific
interaction with Shiga toxin 2 produced by E. coli O157:H7
⇑
Jared M. Jacobson, Pavel I. Kitov, David R. Bundle
Alberta Glycomics Centre, Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 April 2013
Received in revised form 11 May 2013
Accepted 13 May 2013
Available online 28 May 2013
Hemolytic uremic syndrome is a potentially fatal complication of food poisoning caused by Escherichia
coli O157:H7, especially those strains that produce the Stx2 Shiga toxin. Multivalent inhibitors based
on the Pk trisaccharide are most effective against Stx1 the less dangerous of the two Shiga toxins. Inhib-
itors containing a terminal 2-acetamido-2-deoxy-
-galactopyranosyl residue of Pk trisaccharide have been shown to exhibit preferential binding to
Stx2. A multivalent heterobifunctional Pk analog containing 2-acetamido-2-deoxy-
-galactopyranose
a-D-galactopyranosyl residue in place of the terminal
a
-D
a-D
This paper is dedicated to the memory of
Dr. Malcolm Perry and Porfessor Lennart Kenne
has been synthesized in a format that facilitates the ablation of toxin activity via supramolecular complex
formation between Stx and the endogenous protein, Human serum amyloid P component (HuSAP).
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Shiga toxin 2
Pk trisaccharide
Toxin inhibitors
Trisaccharide synthesis
Heterobifunctional ligand
Escherichia coli O157:H7
1. Introduction
interaction.10 We have previously reported the effective inhibition
of Stx1 by a decavalent dendrimer, STARFISH.11 This molecule has
Hemolytic uremic syndrome (HUS) is a potentially fatal kidney
disease caused by Shiga toxin producing E. coli (STEC), such as
E. coli O157:H7 and E. coli O104:H4.1,2 E. coli O157:H7 produces
two types of phage encoded Shiga toxins, Shiga toxin 1 (Stx1)
and Shiga toxin 2 (Stx2) which share approximately 56% sequence
identity.3 Shiga toxins are AB5 holotoxins consisting of a catalytic A
subunit, responsible for the cleavage of a single nucleotide residue
on rRNA resulting in cell death, and the cell surface binding B5
homopentameric subunit.4–6 The natural ligand for Stx1 is
Globotriaosyl ceramide (Gb3) consisting of Pk trisaccharide
a pentameric display of bivalent ligands, which was found to bring
together two molecules of Stx1 in a face-to-face manner to form a
supramolecular complex.11 Expanding upon the principle of face-
to-face aggregation of protein, we later reported the synthesis of
a heterobifunctional ligand, BAIT-Pk, and its polymeric counterpart,
PolyBAIT-Pk, which allowed for the simultaneous binding of Stx1
and an endogenous protein of the innate immune system, Human
serum amyloid P component (HuSAP).12,13 HuSAP acts as a supra-
molecular template protein. Like Stx it is a pentameric, radially
symmetric protein. Additionally, analysis of both the Stx1-B5 sub-
unit and HuSAP indicated that when the two proteins are overlaid,
their respective binding sites can be brought into register. The li-
gand PolyBAIT-Pk was designed to incorporate the Stx1 binding Pk
trisaccharide and the HuSAP binding pyruvate moiety14 to enable
the formation of a supramolecular complex (Fig. 2). In vitro and
in vivo studies of PolyBAIT-Pk showed sequestering of the toxin
and its removal via the liver, thereby affording protection of HuSAP
transgenic mice in vivo after challenge with a lethal dose of Shiga
toxin.13
(a
Gal(1?4)-bGal(1?4)-bGlc) (Fig. 1).7 It has been shown that
Stx1 binds Gb3 with greater affinity than Stx2, an apparent anom-
aly to the observation that Stx2 producing bacteria are more likely
to result in the development of HUS than those producing Stx1.8
However, an alternative receptor for Stx2 other than Gb3, is yet
to be elucidated. Recently Kale et al. have shown that a modified
Pk trisaccharide derivative with a terminal 2-acetamido-2-deoxy-
D
-galactose moiety (Fig. 1) binds Stx2 selectively over Stx1.9
The intrinsic affinity of proteins for carbohydrate ligands is low
and disassociation constants, Kd, often fall in the mM range. This can
be overcome through the use of multivalency or supramolecular
As Stx2 is found to be the most clinically relevant of the tox-
ins,15 we applied our previously established heterobifunctional li-
gand strategy to the development of a Stx2 specific ligand. We
present here the synthesis of a Stx2 specific multivalent heterobi-
functional ligand.
⇑
Corresponding author. Tel.: +1 780 492 8808; fax: +1 780 492 7705.
E-mail address: dave.bundle@ualberta.ca (D.R. Bundle).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.05.010

J. M. Jacobson et al. / Carbohydrate Research 378 (2013) 4–14
5
HO
HO
to provide a para-methoxybenzylidene intermediate which was
subsequently treated with benzoyl chloride in pyridine to provide
the intermediate 13 in 88% over two steps.17 The para-methoxy-
benzylidene acetal of 13 was regioselectively opened using
BH3ꢀTHF and TMSOTf to provide the 6-OH, 4-O-p-methoxybenzyl
intermediate which was acetylated to provide 6 in 84% yield over
two steps.20
OH
O
R
O
OH
O
OH
O
HO
O
OR'
HO
OH
OH
R = OH: Pk trisaccharide
R = NHAc: PkNAc trisaccharide
2.3. Trisaccharide formation
Figure 1. Pk trisaccharide; PkNAc trisaccharide.
The construction of the trisaccharide framework was completed
by glycosidation of thiophenyl donor 6 and the glucosyl acceptor 7
using N-iodosuccinimide (NIS) and AgOTf at ꢁ20 °C to give the lac-
tosyl derivative 14 in 90% yield (Scheme 4).21 The selective depro-
tection of the 40-OPMB ether was done by the addition of one
equivalent of TfOH at ꢁ20 °C to give the lactosyl 40-OH acceptor
15 in 81% yield. Subsequent glycosylation of acceptor 15 by trichlo-
roacetimidate donor 522 was performed using TMSOTf in Et2O at
room temperature to provide the trisaccharide 16 in a modest
yield, 48%. Decomposition of the donor was observable before
the formation of the glycosidic linkage and often resulted in no
yield of the product. This problem was circumvented by the slow
drop-wise addition of donor to a mixture of acceptor and activa-
tor.23 The azide 16 was reduced to the corresponding amine using
dithiothreitol (DTT) in a mixture of CH3CN:Et3N (6:1)24 and con-
verted to the N-acetyl derivative after treatment with Ac2O and
pyridine to give 17 in 87% yield. The benzyl ether was selectively
removed by hydrogenation to give 3 in 97% yield.
2. Results and discussion
A retrosynthetic analysis of 1 requires the incorporation of four
necessary moieties (Scheme 1); a linker 4 that allows for conjuga-
tion to a polymeric scaffold and three monosaccharide building
blocks, 5, 6, and 7.
The synthesis of 1 was accomplished by click reaction between
BAIT-PkNAc, 2 and an azido-polyacrylamide. BAIT-PkNAc 2 was
synthesized via coupling of the amine linker 4 and a Pk trisaccha-
ride derivative obtainable from 3. The Pk trisaccharide derivative
is synthesized via the coupling of the thioglycoside donor 6 with
acceptor 7, which after glycosylation with trichloroacetimidate do-
nor 5 provides the Pk trisaccharide derivative 3.
2.1. Synthesis of acceptor 7
2.4. Completion of PolyBAIT-PkNAc 1
The synthesis of the acceptor 7 was accomplished via the for-
mation of the known cyanoethylidene derivative 8 from the corre-
sponding glycosyl bromide according to the literature procedures
(Scheme 2).16 The fully acetylated cyanoethylidene 8 underwent
global deprotection using NaOCH3 in CH3OH to first remove all
the acetate groups and to aid in the formation of the methoxy(eth-
ylidene) moiety, by forming the corresponding methoxy imidate
species 8a. Treatment of 8a with glacial CH3COOH results in the
To complete the synthesis of 1, intermediate 3 was combined
with 4-nitrophenyl chloroformate in the presence of pyridine to
make the 4-nitrophenyl carbonate 18 in 99% yield (Scheme 5).
Treatment of 18 with amine 4 under basic conditions gave 19 in
95% yield. Global deprotection of 19 was achieved through the
use of one equivalent of 1 M NaOCH3 in CH3OH to remove ester
groups. Subsequent treatment with water, converted the methyl
ester of the pyruvate moiety to the corresponding carboxylic acid,
to form 2 in 88% yield over two steps. Lastly, a Cu catalyzed [3+2]-
Huisgen cycloaddition25,26 was performed to conjugate the mono-
meric ligand 2 to the previously synthesized poly[acrylamide-co-
(3-azidopropylmethacrylamide)]27 with polydispersity index
(PDI) of 1.29 to provide the desired PolyBAIT-PkNAc product 1.
formation of
9 in 75% yield. The triol 9 was treated with
PhCH(OCH3)2 and camphorsulfonic acid (CSA) to form the 4,6-O-
benzylidene intermediate and subsequent acetylation of the
remaining 3-OH gave the benzylidene derivative 10 in 85% yield
over two steps.17 Lastly, the benzylidene acetal 10 was regioselec-
tively opened using Et3SiH and trifluoroacetic acid (TFA) to provide
the selectively protected glycosyl acceptor 7 in 85% yield.18
2.2. Synthesis of donor 6
2.5. Conclusion
The synthesis of the thiophenyl galactoside donor 6 was
achieved through the synthesis of the known thiogalactoside 12
from the peracetylated derivative 1119 using NaOCH3 in CH3OH
to give 12 in 97% yield (Scheme 3). The deprotected galactoside
12 was treated with the dimethyl acetal of anisaldehyde and CSA
We have described the synthesis of a Stx2 specific multivalent
heterobifunctional ligand, PolyBAIT-PkNAc. This approach differs
from previous syntheses of PolyBAIT-Pk where our approach was
to employ a linking arm terminated with a pentenyl group for co
polymerization with acrylamide. The adoption of the azide/alkyne
Figure 2. Left: PolyBAIT-Pk. Right: Graphical representation of a supramolecular complex: Yellow = HuSAP, Blue = Stx1, and Polymer = PolyBAIT-Pk.

6
J. M. Jacobson et al. / Carbohydrate Research 378 (2013) 4–14
HO
HO
OH
O
H
N
O
AcHN
H NOC
O
2
OH
O
O
O
O
O
H NOC
2
O
O
HO
O
N
H
H
N
HO
O
OH
O
N
O
N
N
H NOC
2
COOH
H NOC
2
1
HO
HO
OH
O
H
O
N
H NOC
2
AcHN
HO
O
OH
O
O
O
H NOC
2
O
O
H
N
O
O
O
N
H
HO
O
OH
N
3
O
O
H NOC
2
COOH
H NOC
2
2
Aza-polyacrylamide
AcO
AcO
OAc
O
H N
2
AcHN
O
O
OAc
O
O
N
O
OH
O
BzO
O
O
H
AcO
OBz
O
O
4
COOCH
3
3
OBn
O
AcO
AcO
OAc
O
PMBO
BzO
OAc
O
HO
AcO
O
O
SPh
CCl
3
O
N
3
OBz
HN
COOCH
3
5
6
7
Scheme 1. Retrosynthetic pathway to achieve synthesis of PolyBAIT-PkNAc 1.
cycloaddition provides a more flexible approach to the creation of
polymeric inhibitors, which we have also applied to other poly-
meric scaffolds that display Stx ligands. In vitro and in vivo results
are currently being obtained and will be presented elsewhere.
under argon atmosphere. Molecular sieves were dried in an oven
maintaining an internal temperature of 350 °C to ensure dryness
and were allowed to cool under vacuum or under argon atmo-
sphere at room temperature. Reactions were monitored by analyt-
ical thin-layered chromatography (TLC) on Silica gel 60-F254 (E.
Merck). Plates were visualized under UV light and/or by treatment
with either 5% sulfuric acid in ethanol, potassium permanganate
solution, ninhydrin solution, or molybdate solution followed by
charring 250 °C. All solvents were removed by rotary evaporation
at <40 °C unless otherwise stated. Flash column chromatography
was performed using silica gel (230–400 mesh, Silicycle, Montreal)
3. Experimental
3.1. General methods
All chemical reagents obtained were of analytical grade and
used as obtained from commercial sources unless otherwise indi-
cated. Solvents used in water-sensitive reactions were purified by
passage through columns of alumina and copper under nitrogen
atmosphere except for methanol, which was distilled prior to use
over magnesium methoxide and collected as needed. Unless other-
wise stated, all reactions were performed at room temperature and
at flow rates between 6 and 18.5 mL minꢁ1 1H NMR spectra were
.
recorded at 500, 600, or 700 MHz. Chemical shifts are reported in
ppm (d) and were referenced to internal residual protonated sol-
vent signals or to external acetone in the case of D2O (0.1% external
acetone d 2.225 ppm). 13C NMR spectra were recorded at 125 MHz

J. M. Jacobson et al. / Carbohydrate Research 378 (2013) 4–14
7
OH
O
OAc
O
O
Ph
O
O
O
HO
AcO
AcO
AcO
a
b
HO
O
O
O
O
O
COOCH
3
COOCH
CN
3
8
9
10
OH
O
OBn
O
HO
HO
HO
AcO
c
O
O
O
O
OCH
3
COOCH
3
HN
7
8a
Scheme 2. Reagents and conditions: (a) (i) NaOCH3, CH3OH, (ii) CH3COOH, 75%; (b) (i) PhCH(OCH3)2, CSA, CH3CN, (ii) Ac2O, Pyr, 83%; and (c) Et3SiH, TFA, CH2Cl2, 0 °C, 85%.
OMe
O
AcO
AcO
HO
HO
OAc
O
OH
O
a
b
O
SPh
SPh
O
SPh
BzO
OAc
11
OH
12
OBz
13
PMBO
BzO
OAc
O
c
SPh
OBz
6
Scheme 3. Reagents and conditions: (a) NaOCH3, CH3OH, 97%; (b) (i) CH3OPhCH(OCH3)2, CSA, CH3CN, 30 °C, (ii) BzCl, pyridine, 88%; (c) (i) BH3ꢀTHF, TMSOTf, (ii) Ac2O,
pyridine, 84%.
and chemicals shifts are referenced to internal CDCl3 (77.23 ppm)
or external acetone (31.07 ppm). High resolution mass spectra
were obtained on a Micromass Zabspec TOF-mass spectrometer
by the analytical services of this Department. Optical rotations
were determined with a Perkin–Elmer model 241 polarimeter at
room temperature using the sodium D-line and are reported in
deg mL gꢁ1 dmꢁ1. Combustion analysis was performed by analyti-
cal services of this Department.
hexanes to provide the pure exo-CN product 8 (7.26 g, 42%) as a
white needle solid; Rf 0.52 (1:1, hexanes–ethyl acetate); [
a
]
D
+12.9° (c 1.01, CHCl3); 1H NMR (500 MHz, CDCl3) d 5.80 (d, 1H,
J1,2 5.1 Hz, H-1), 5.21 (vt, 1H, J2,3 2.8 Hz, J3,4 2.8 Hz, H-3), 4.91
(ddd, 1H, J2,4 0.8 Hz, J2,4 2.6 Hz, J4,5 9.6 Hz, H-4), 4.39 (ddd, 1H,
J2,4 0.9 Hz, J2,3 2.9 Hz, J1,2 5.2 Hz, H-2), 4.20 (m, 1H, H-6a), 4.19
(m, 1H, H-6b), 3.90 (ddd, 1H, J5,6a 4.3 Hz, J5,6b 4.3 Hz, J4,5 9.3 Hz,
H-5), 2.14 (s, 3H, CH3C(O)O), 2.09 (s, 3H, CH3C(O)O), 2.09 (s, 3H,
CH3C(O)O), 1.92 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3) d 170.6
(CH3C(O)O), 169.5 (CH3C(O)O), 168.9 (CH3C(O)O), 116.4 (C„N),
98.8 (quat. C pyruvate), 97.4 (C-1), 74.2 (C-2), 69.3 (C-3), 67.8 (C-
4), 67.3 (C-5), 62.8 (C-6), 24.3 (–CH3), 20.7 (CH3C(O)O); ESI HRMS:
calcd for C15H19NO19Na 380.0952. Found 380.0947; Anal. Calcd for
3.1.1. 3,4,6-Tri-O-acetyl-1,2-O-[(S)-1-(cyano)ethylidene]-a-D-
glucopyranoside (8)
Per-O-acetyl-a-D-glucopyranosyl bromide (19.9 g, 0.0485 mol)
was combined with ground up KCN (15.8 g 0.242 mol) and TBAB
(5.10 g, 0.0242 mol) in a round bottomed flask to which dry aceto-
nitrile (120 mL) was added. The vessel was placed under argon
atmosphere and the reaction proceeded at room temperature for
3 days. The reaction turned a dark brown color, indicating a com-
pleted reaction. Completion of the reaction was confirmed using
1H NMR spectroscopy, which showed no remaining anomeric pro-
ton signal for the glucosyl bromide. The reaction mixture was then
filtered through Celite and concentrated to dryness. The crude
reaction mixture was then subjected to flash column chromatogra-
phy on silica gel using 2:1, hexanes–ethyl acetate. Fractions were
collected and found to contain both exo-CN and endo-CN products.
The exo-CN isomer was recrystalized from ethyl acetate and
C15H19NO19: C, 50.42; H, 5.36; N, 3.92. Found: C, 50.59; H, 5.33; N,
3.93.
3.1.2. 1,2-O-[(S)-1-(Methoxycarbonyl)ethylidene]-a-D-
glucopyranoside (9)
Cyanoethylidene 8 (6.56 g, 18.4 mmol) was dissolved in anhy-
drous methanol (60 mL). The flask was placed under argon atmo-
sphere and sodium metal (0.20 g) was added and allowed to
dissolve. The reaction was allowed to proceed for 2 days at which
point TLC analysis (20%, methanol–dichloromethane) showed no
remaining starting material. Glacial acetic acid (40 mL) was added
and the reaction proceeded overnight. The reaction mixture was

8
J. M. Jacobson et al. / Carbohydrate Research 378 (2013) 4–14
PMBO
BzO
OAc
O
OBn
O
OBn
O
PMBO
BzO
OAc
O
HO
a
O
AcO
AcO
OBz
O
SPh
O
O
O
OBz
COOCH
3
COOCH
6
3
7
14
R = OPMB
b
15
R = OH
AcO
AcO
OAc
O
NH
CCl
c
N
O
3
3
5
AcO
AcO
AcO
AcO
OAc
O
OAc
O
N
AcHN
3
O
O
OAc
O
OAc
O
d
OR
O
OBn
O
O
O
BzO
BzO
AcO
OBz
AcO
OBz
O
O
O
O
COOCH
COOCH
3
3
17
16
R = OBn,
e
3
R = OH,
Scheme 4. Reagents and conditions: (a) NIS, AgOTf, 4 Å MS, CH2Cl2, ꢁ20 °C, 90%; (b) TfOH, CH2Cl2, ꢁ20 °C, 81%; (c) 15, TMSOTf, 4 Å MS, Et2O, 48%; (d) (i) DTT, CH3CN:Et3N
(6:1), (ii) Ac2O, Pyr 87%; and (e) H2, Pd(OH)2/C, 97%.
subsequently co-evaporated with toluene. Once dry, the solid was
dissolved in methanol, and adsorbed onto silica gel and purified by
flash column chromatography (5%, methanol–dichloromethane) to
(dd, 1H, J2,3 3.5 Hz, J3,4 3.5 Hz, H-3), 4.41 (dd, 1H, J5,6a 5.2 Hz,
J6a,6b 10.6 Hz, H-6a), 4.33 (dd, 1H, J2,3 3.4 Hz, J1,2 5.1 Hz, H-2),
3.94 (ddd, J5,6a 5.3 Hz, J4,5 9.8 Hz, H-5), 3.77 (s, 3H, CH3OC(O)),
3.72 (m, 2H, H-4, H-6b), 2.12 (s, 3H, CH3C(O)O), 1.76 (s, 3H, CH3);
13C NMR (125 MHz, CDCl3): d 169.8 (C@O), 169.5 (C@O), 136.8
(Ar), 129.2 (Ar), 128.3 (Ar), 126.1 (Ar), 104.0 (quaternary C-pyru-
vate), 101.6 (PhCH benzylidene), 98.8 (C-1), 77.7 (C-4), 77.2 (C-
2), 73.1 (C-3), 68.8 (C-6), 62.3 (C-5), 52.7 (C(O)OCH3), 22.4
(CH3C(O)O), 20.9 (CH3 pyruvate); ESI HRMS calcd for C19H22O9Na
417.1156. Found 417.1154; Anal. Calcd for C19H22O9: C, 57.86; H,
5.62. Found: C, 58.01; H, 5.77.
give the product 9 (3.48 g, 72%) as a white solid; [a]D +34.5° (c 1.10,
CH3OH); 1H NMR (500 MHz, D2O) d 5.77 (d, 1H, J1,2 4.9 Hz, H-1),
4.27 (ddd, 1H, J2,4 0.8 Hz, J2,3 2.2 Hz, J1,2 5.0 Hz, H-2), 3.99 (vt, 1H,
J2,3 4.0 Hz, J3,4 4.0 Hz, H-3), 3.85 (m, 1H, H-6a), 3.80 (s, 3H,
COOCH3), 3.72 (m, 2H, H-5, H-6b), 3.58 (ddd, 1H, J2,4 0.7 Hz, J3,4
3.9 Hz, J4,5 9.1 Hz, H-4), 1.72 (s, 3H, CH3 (pyruvate)); 13C NMR
(125 MHz, D2O): d 172.2 (C(O)OCH3), 105.7 (quart. C pyruvate),
98.6 (C-1), 76.9 (C-2), 73.7 (C-5), 72.4 (C-3), 69.0 (C-4), 62.2 (C-
6), 54.3 (C(O)OCH3), 22.2 (CH3 pyruvate); ESI HRMS Calcd for
C10H16O8Na 287.0737. Found 287.0733.
3.1.4. 3-O-Acetyl-6-O-benzyl-1,2-O-[(S)-1-
(methoxycarbonyl)ethylidene]-a-D-glucopyranoside (7)
3.1.3. 3-O-Acetyl-4,6-O-benzylidene-1,2-O-[(S)-1-
(methoxycarbonyl)ethylidene]- -glucopyranoside (10)
Glucoside 9 (2.58 g, 9.76 mmol) was dissolved in anhydrous
acetonitrile (30 mL). -Dimethoxytoluene (1.97 mL, 13.6 mmol)
Benzylidene acetal 10 (3.89 g, 9.88 mmol) was dissolved in dry
dichloromethane (20 mL) and the reaction vessel placed under an
argon atmosphere. Et3SiH (16 mL, 98.8 mmol) was added and the
mixture cooled to 0 °C using an ice-water bath. TFA (7.6 mL,
98.8 mmol) was added and the reaction allowed to proceed at
0 °C. After 1 h, TLC analysis (15%, ethyl acetate–toluene) showed
no remaining starting material. The reaction mixture was diluted
with dichloromethane and transferred to a separatory funnel
where the organic layer was washed with saturated aqueous so-
dium bicarbonate, distilled water, and saturated aqueous sodium
chloride. The organic layer was dried over anhydrous sodium sul-
fate, filtered, and concentrated to dryness. The crude product was
purified by flash column chromatography on silica gel (15% ?
30%, ethyl acetate–toluene) to provide the product 7 (3.35 g, 85%)
a-D
a,a
was then added followed by a catalytic amount of CSA (20 mg).
The reaction was carried out at reduced pressure at 30 °C to re-
move methanol generated during reaction. After 2.5 h, TLC analysis
(5%, methanol–dichloromethane) showed that the reaction was
complete. The reaction was quenched with five drops of Et3N and
concentrated. The crude acetal product was dissolved in pyridine
(50 mL) and acetic anhydride (50 mL) and the reaction allowed to
proceed overnight under argon atmosphere after which TLC analy-
sis (2:1, hexanes–ethyl acetate) showed maximum product forma-
tion. The mixture was co-evaporated with toluene and purified by
flash column chromatography on silica gel (3:1 ? 1:1, hexanes–
ethyl acetate) to provide the product 10 (3.18 g, 83%) as a white so-
as a white solid; Rf 0.18 (15%, ethyl acetate–toluene); [a] +12.9°
D
(c 1.03, CHCl3); 1H NMR (500 MHz, CDCl3) d 7.35–7.26 (m, 5H,
ArH), 5.81 (d, 1H, J1,2 5.1 Hz, H-1), 5.03 (vt, 1H, J2,3 3.4 Hz, J3,4
3.4 Hz, H-3), 4.62 (d, 1H, Jgem 12.2 Hz, PhCH2O), 4.57 (d, 1H, Jgem
12.1 Hz, PhCH2O), 4.38 (ddd, 1H, J2,4 0.8 Hz, J2,3 3.1 Hz, J1,2 5.1 Hz,
lid; Rf 0.56 (1:1, hexanes–ethyl acetate); [a]D +27.1° (c 1.02, CHCl3);
1H NMR (500 MHz, CDCl3) d 7.47 (m, 2H, ArH), 7.37 (m, 3H, ArH),
5.84 (d, 1H, J1,2 5.1 Hz, H-1), 5.53 (s, 1H, C–H benzylidene), 5.24

J. M. Jacobson et al. / Carbohydrate Research 378 (2013) 4–14
9
AcO
AcO
AcO
AcO
OAc
O
OAc
O
O
O
AcHN
AcHN
O
O
OAc
O
OAc
O
a
OH
O
O
O
O
O
O
BzO
BzO
AcO
AcO
NO
2
OBz
OBz
O
O
O
COOCH
COOCH
3
3
3
18
O
H N
2
O
b
O
N
H
O
4
AcO
AcO
OAc
O
H
N
O
AcHN
BzO
O
OAc
O
c
O
O
O
O
O
O
AcO
N
H
O
OBz
O
O
COOCH
3
19
HO
HO
OH
O
H
N
O
AcHN
O
OH
O
d
O
O
k
O
O
1, PolyBAIT-P NAc
O
O
O
HO
N
HO
O
H
OH
O
COOH
2
Scheme 5. Reagents and conditions: (a) 4-nitrophenyl chloroformate, pyr, CH2Cl2, 99%; (b) amine 4, Et3N, CH2Cl2, 95%; (c) (i) 1 M NaOCH3, CH3OH, (ii) H2O, 88%; (d) aza-
polyacrylamide, sodium ascorbate, CuSO4, and H2O.
H-2), 3.83 (m, 1H, H-5), 3.76 (m, 4H, H-4, CH3OC(O)), 3.71 (m, 2H,
H-6a, H-6b), 2.83 (br s, 1H, O-H), 2.09 (s, 3H, CH3C(O)O), 1.77 (s,
3H, CH3); 13C NMR (125 MHz, CDCl3): d 170.7 (C@O), 169.4
(C@O), 137.7 (Ar), 128.4 (Ar), 127.8 (Ar), 104.9 (quaternary C pyru-
vate), 98.1 (C-1), 74.8, 74.5 (C-2, C-3), 73.7 (PhCH2O), 70.2 (C-5),
69.6 (C-6), 69.2 (C-4), 52.7 (C(O)OCH3), 21.7 (CH3C(O)O), 20.9
(CH3 pyruvate); ESI HRMS calcd for C19H24O9Na 419.1313. Found
419.1311; Anal. Calcd for C19H24O9: C, 57.57; H, 6.10. Found: C,
57.29; H, 6.45.
(1:1, hexanes–ethyl acetate) showed maximum product formation.
The reaction was quenched with Et3N until neutral and the mixture
was diluted with dichloromethane (20 mL) and concentrated to
dryness. The crude product was purified by flash column chroma-
tography on silica gel (2:1, hexanes–ethyl acetate) to obtain the
product 11 (45.1 g, 98%) as a white foam; Rf 0.40 (1:1, hexanes–
ethyl acetate); [a]
D + 3.5° (c 1.06, CHCl3); 1H NMR (500 MHz,
CDCl3) d 7.52 (m, 2H, ArH), 7.32 (m, 3H, ArH), 5.42 (dd, 1H, J4,5
1.0 Hz, J3,4 3.4 Hz, H-4), 5.24 (dd, 1H, J2,3 9.9, J1,2 9.9 Hz, H-2),
5.06 (dd, 1H, J3,4 3.3 Hz, J2,3 9.9 Hz, H-3), 4.72 (d, 1H, J1,2 9.9 Hz,
H-1), 4.20 (dd, 1H, J5,6a 7.0 Hz, J6a,6b 11.3 Hz, H-6a), 4.12 (dd, 1H,
J5,6b 6.2 Hz, J6a,6b 11.4 Hz, H-6b), 3.94 (ddd, 1H, J4,5 1.0, J5,6b
6.1 Hz, J5,6a 7.1 Hz, H-5), 2.12 (s, 3H, CH3C(O)O), 2.10 (s, 3H,
CH3C(O)O), 2.05 (s, 3H, CH3C(O)O), 1.98 (s, 3H, CH3C(O)O); 13C
3.1.5. Phenyl 2,3,4,6-tetra-O-acetyl-1-thio-b-D-
galactopyranoside (11)
a,b-Galactose pentaacetate (40.6 g, 0.104 mol) was dissolved in
dry dichloromethane (60 mL) and placed under argon atmosphere.
PhSH (16.0 mL, 0.156 mol) was added and the solution was cooled
to 0 °C in an ice-water bath while stirring. Once cool BF3ꢀOEt2,
(19.6 mL, 0.156 mol) was added and the reaction allowed to pro-
ceed while warming to room temperature. After 2 h TLC analysis
NMR (125 MHz, CDCl3):
d 170.3 (C@O), 170.2 (C@O), 170.0
(C@O), 169.4 (C@O), 132.6 (Ar), 132.4 (Ar), 128.9 (Ar), 128.1
(C@O), 86.6 (C-1), 74.4 (C-5), 72.0 (C-3), 67.3, 67.2 (C-2, C-4),
61.6 (C-6), 20.8 (CH3C(O)O), 20.6 (CH3C(O)O), 20.5 (CH3C(O)O);

10
J. M. Jacobson et al. / Carbohydrate Research 378 (2013) 4–14
ESI HRMS calcd for C20H24O9SNa 463.10332. Found 463.10322;
Anal. Calcd for C20H24O9S: C, 54.54; H, 5.49; S, 7.28. Found: C,
54.38; H, 5.56; S, 7.29.
temperature for 1.5 h at which point TLC (1:1, hexanes–ethyl ace-
tate) showed no remaining starting material. The reaction was
quenched with two drops of Et3N followed by very slow addition
of methanol until bubbling ceased. The reaction mixture was then
concentrated to dryness and subsequently dissolved in a 1:1 mix-
ture of pyridine and acetic anhydride (10 mL) and allowed to react
overnight. TLC analysis (1:1, hexanes–ethyl acetate) showed no
remaining intermediate. The mixture was co-evaporated with tol-
uene (3 ꢂ 20 mL) until dry. The crude product was purified by flash
column chromatography on silica gel (3:1 ? 2:1, hexanes–ethyl
acetate) and recrystalized from ethyl acetate and hexanes to give
the product 6 (0.486 g, 84%) as a white powder; Rf 0.58 (1:1, hex-
3.1.6. Phenyl 1-thio-b-D-galactopyranoside (12)
The acetylated thiogalactoside 11 (11.84 g, 26.9 mmol) was dis-
solved in 60 mL anhydrous methanol to which a catalytic amount
of sodium metal (0.045 g) was added. The reaction was allowed to
proceed overnight at room temperature and under argon atmo-
sphere. Subsequent TLC analysis (20%, methanol–dichlorometh-
ane) showed no remaining starting material The reaction was
neutralized with Dowex H+ ion exchange resin, filtered, and con-
centrated to dryness. The crude product was recrystalized from
anhydrous ethanol and filtered to give the pure product 12
(7.10 g, 97%) as a white solid; Rf 0.79 (20%, methanol–dichloro-
anes–ethyl acetate); [a]
+66.7° (c 1, CHCl3); 1H NMR (500 MHz,
D
CDCl3) d 7.96 (m, 4H, ArH), 7.51 (m, 4H, ArH), 7.38 (m, 4H, ArH),
7.26 (m, 3H, ArH), 7.13 (m, 2H, ArH), 6.76 (m, 2H, ArH), 5.87 (vt,
1H, J1,2 10.0 Hz, J2,3 10.0 Hz, H-2), 5.34 (dd, 1H, J3,4 2.9 Hz, J2,3
10.0 Hz, H-3), 4.91 (d, 1H, J1,2 10.0 Hz, H-1), 4.68 (d, 1H, Jgem
11.4 Hz, CH3OPhCH2O), 4.46 (d, 1H, Jgem 11.4 Hz, OCH2PhOCH3),
4.35 (dd, 1H, J5,6a 6.8 Hz, J6a,6b 11.3 Hz, H-6a), 4.16 (d, 1H, J3,4
2.8 Hz, H-4), 4.11 (dd, 1H, J5,6b 6.2 Hz, J6a,6b 11.3 Hz, H-6b), 3.91
(m, 1H, H-5), 3.76 (s, 3H, OCH2PhOCH3), 2.03 (s, 3H, CH3C(O)O);
13C NMR (125 MHz, CDCl3) d 170.4 (C@O), 165.9 (C@O), 165.2
(C@O), 159.3 (ArC-O), 133.5 (Ar), 133.2 (Ar), 132.8 (Ar), 132.5
(Ar), 129.9 (Ar), 129.8 (Ar), 129.5 (Ar), 129.4 (Ar), 128.9 (Ar),
128.8 (Ar), 128.5 (Ar), 128.4 (Ar), 127.9 (Ar), 113.8 (Ar), 86.7 (C-
1), 76.1 (C-5), 75.9 (C-3), 74.3 (CH3OPhCH2O), 72.9 (C-4), 68.3 (C-
2), 62.7 (C-6), 55.2 (CH3OPhCH2O), 20.8 (CH3C(O)O); ESI HRMS
calcd for C36H34O9SNa 665.1816. Found 665.1806; Anal. Calcd for
methane); [
a
]
ꢁ51.6° (c 1.00, CH3OH); 1H NMR (500 MHz, D2O)
D
d 7.65 (m, 2H, ArH), 7.47 (m, 3H, ArH), 4.84 (d, 1H, J1,2 13.2 Hz,
H-1), 4.06 (d, 1H, J3,4 3.2 Hz, H-4), 3.85–3.75 (m, 4H, H-3, H-5, H-
6ab), 3.70 (m, 1H, H-2); 13C NMR (125 MHz, D2O): d 133.7 (Ar),
132.1 (Ar), 130.3 (Ar), 128.8 (Ar), 89.0 (C-1), 79.9 (C-5), 74.9 (C-
3), 70.2 (C-2), 69.7 (C-4), 61.9 (C-6); ESI HRMS calcd for
C12H16O5Na 295.0611. Found 295.0607; Anal. Calcd for C12H16O5:
C, 52.93; H, 5.92; S, 11.77. Found: C, 52.67; H, 5.99; S, 11.76.
3.1.7. Phenyl 2,3-di-O-benzoyl-4,6-O-para-
methoxybenzylidenyl-1-thio-b-
D
-galactopyranoside (13)
-galactopyranoside 12
To a solution of dry phenyl 1-thio-b-
D
(5.43 g, 19.9 mmol) in anhydrous acetonitrile (50 mL), CH3OPh-
CH(OCH3)2 (4.1 mL, 23.9 mmol) was added followed by a catalytic
amount of CSA (100 mg). The reaction was done at reduced pres-
sure at 30 °C to remove methanol generated during the reaction.
After 1.5 h, TLC analysis (10%, methanol–dichloromethane) showed
the reaction to be complete. The reaction was quenched with five
drops of Et3N and concentrated. The crude para-methoxybenzyli-
dene product was dissolved in pyridine (50 mL) and BzCl
(11.6 mL, 99.7 mmol) was added slowly and the reaction pro-
ceeded overnight. Once again TLC analysis (2:1, hexanes–ethyl ace-
tate) showed maximum product formation and no remaining
starting material. The mixture was concentrated to dryness while
co-evaporating with toluene (3 ꢂ 20 mL). The crude product was
purified by flash column chromatography on silica gel (3:1 ?
1:1, hexanes–ethyl acetate) to provide the product 13 (10.5 g,
C36H34O9S: C, 67.27; H, 5.33; S, 4.99. Found: C, 67.37; H, 5.33; S,
4.78.
3.1.9. 6-O-acetyl-2,3-di-O-benzoyl-4-O-para-methoxybenzyl-b-
D-galactopyranosyl-(1?4)-3-O-acetyl-6-O-benzyl-1,2-O-[(S)-1-
(methoxycarbonyl)ethylidene]- -glucopyranoside (14)
a-D
The acceptor 7 (0.518 g, 1.31 mmol) and donor 6 (1.26 g,
1.96 mmol) were combined with pre-activated 4 Å molecular
sieves (0.500 g) and dissolved in anhydrous dichloromethane
(25 mL). The mixture was stirred for 1 h at room temperature
and under argon atmosphere. The contents were cooled to
ꢁ20 °C after which the reaction was activated by the addition of
NIS (0.209 g, 1.57 mmol) and AgOTf (0.050 g, 0.196 mmol). Reac-
tion progress was monitored by TLC analysis (1:1, hexanes–ethyl
acetate) and after 1 h the reaction was found to be complete. The
reaction was quenched by the addition of Et3N (1 drop) and the
mixture was diluted with dichloromethane. This mixture was fil-
tered through Celite and subsequently washed with saturated
aqueous sodium bicarbonate, saturated aqueous sodium thiosul-
fate, distilled water, and saturated aqueous sodium chloride. The
organic layer was dried over anhydrous sodium sulfate, filtered,
and concentrated to dryness. The crude product was purified by
flash column chromatography on silica gel (1:1, hexanes–ethyl
acetate) to give the product 14 (1.09 g, 90%) as a white foam; Rf
88%) as a white foam; Rf 0.71 (1:1, hexanes–ethyl acetate); [a]
D
+81.4° (c 1, CHCl3); 1H NMR (500 MHz, CDCl3) d 7.97 (m, 2H,
ArH), 7.92 (m, 2H, ArH), 7.62 (m, 2H, ArH), 7.52 (m, 1H, ArH),
7.46 (m, 1H, ArH), 7.39 (m, 2H, ArH), 7.31 (m, 7H, ArH), 6.87 (m,
2H, ArH), 5.80 (t, 1H, J1,2 9.9 Hz, J2,3 9.9 Hz, H-2), 5.46 (s, 1H, ben-
zylidene C–H), 5.35 (dd, 1H, J3,4 3.4 Hz, J3,2 10.0 Hz, H-3), 4.96 (d,
1H, J1,2 9.8 Hz, H-1), 4.57 (d, 1H, J3,4 3.3 Hz, H-4), 4.43 (dd, 1H,
J5,6a 1.3 Hz, J6a,6b 12.3 Hz, H-6a), 4.08 (dd, 1H, J5,6b 1.4 Hz, J6a,6b
12.3, H-6b), 3.82 (s, 3H, CH3OPh), 3.74 (s, 1H, H-5); 13C NMR
(125 MHz, CDCl3) d 166.2 (C@O), 164.9 (C@O), 160.1 (Ar), 133.9
(Ar), 133.3 (Ar), 133.1 (Ar), 131.1 (Ar), 130.2 (Ar), 129.9 (Ar),
129.8 (Ar), 129.7 (Ar), 129.1 (Ar), 128.8 (Ar), 128.4 (Ar), 128.3
(Ar), 128.2 (Ar), 127.8 (Ar), 114.7 (Ar), 113.5 (Ar), 100.9 (benzyli-
dene C–H), 85.3 (C-1), 74.1 (C-3), 73.7 (C-4), 69.9 (C-5), 69.1 (C-
6), 67.1 (C-2), 55.3 (OCH3); ESI HRMS Calcd for C34H30O8SNH4
616.2000. Found 616.1998; Anal. Calcd for C34H30O8S: C, 68.21;
H, 5.05; S, 5.36. Found: C, 68.25; H, 5.28; S, 5.36.
0.38 (1:1, hexanes–ethyl acetate); [a]
+23.9° (c 1.08, CHCl3); 1H
D
NMR (500 MHz, CDCl3) d 7.96 (m, 2H, ArH), 7.89 (m, 2H, ArH),
7.50 (m, 2H, ArH), 7.35 (m, 7H, ArH), 7.19 (m, 2H, ArH), 7.15 (m,
0
0
2H, ArH), 6.75 (m, 2H, ArH), 5.77 (m, 2H, J1,2 4.9 Hz, J1 ,2 7.9 Hz,
J2 ,3 10.4 Hz, H-1, H-20), 5.52 (vt, 1H, J2,3 2.5 Hz, J3,4 2.5 Hz, H-3),
0
0
5.22 (dd, 1H, J3 ,4 3.1 Hz, J2 ,3 10.5 Hz, H-30), 4.65 (m, 2H, Jgem
0
0
0
0
11.3 Hz, J1 ,2 7.9 Hz, CH3OPhCH2O, H-10), 4.45 (d, 1H, Jgem 11.5 Hz,
CH3OPhCH2O), 4.36 (d, 1H, Jgem 12.2 Hz, PhCH2O), 4.31 (ddd, 1H,
0
0
0
0
3.1.8. Phenyl 6-O-acetyl-2,3-di-O-benzoyl-4-O-para-
J2,4 1.0 Hz, J2,3 2.9 Hz, J1,2 5.2 Hz, H-2), 4.27 (dd, 1H, J5 ,6a 5.9 Hz,
J6a ,6b 11.0 Hz, H-6a0), 4.15 (d, 1H, Jgem 12.2 Hz, PhCH2O), 4.11 (m,
2H, H-40, H-6b0), 3.89 (m, 1H, H-4), 3.82 (m, 1H, H-50), 3.74 (s,
3H, CH3OPhCH2O), 3.74 (s, 3H, C(O)OCH3), 3.70 (m, 1H, H-5), 3.39
(m, 2H, J5,6a 2.3 Hz, J6a,6b 10.9 Hz, J5,6b 3.5 Hz, J6a,6b 10.9 Hz, H-6a,
H-6b), 2.07 (s, 3H, CH3C(O)O), 2.01 (s, 3H, CH3C(O)O), 1.67 (s, 3H,
0
0
methoxybenzyl-1-thio-b-D-galactopyranoside (6)
The p-methoxylbenzylidene acetal 13 (0.540 g, 0.902 mmol)
was dissolved in dry dichloromethane (30 mL) to which 1.0 M
BH3ꢀTHF (4.51 mL, 4.51 mmol) was added followed by TMSOTf
(24 lL, 0.135 mmol). The reaction was allowed to proceed at room

J. M. Jacobson et al. / Carbohydrate Research 378 (2013) 4–14
11
CH3); 13C NMR (125 MHz, CDCl3) d 170.4 (C@O), 169.5 (C@O),
169.2 (C@O), 165.9 (C@O), 164.9 (C@O), 159.4 (ArOCH3), 137.9
(Ar), 133.5 (Ar), 133.2 (Ar), 130.1 (Ar), 129.8 (Ar), 129.6 (Ar),
129.5 (Ar), 129.4 (Ar), 128.9 (Ar), 128.5 (Ar), 128.4 (Ar), 128.4
(Ar), 127.7 (Ar), 113.8 (Ar), 105.3 (C, pyruvate), 102.6 (C-10), 98.1
(C-1), 76.1 (C-4), 74.6 (C-2), 74.5 (CH3OPhCH2O), 74.1 (C-30), 73.1
(PhCH2O), 72.6, 72.3 (C-40, C-50), 70.6 (C-3), 69.9 (C-20), 68.6 (C-
5), 68.3 (C-6), 62.0 (C-60), 55.2 (CH3OPhCH2O), 52.6 (C(O)OCH3),
21.2 (CH3C(O)O), 20.9 (CH3C(O)O), 20.8 (CH3, pyruvate); ESI HRMS
calcd for C55H58O18Na 917.3871. Found 917.3864; Anal. Calcd for
at which point the donor was consumed. The reaction was
quenched with one drop of Et3N, filtered through Celite, and con-
centrated to dryness. The crude reaction mixture was purified by
flash column chromatography in silica gel (1:1, ethyl acetate–hex-
anes) to provide the product 16 (0.134 g, 48%) as a white foam; Rf
0.44 (60%, ethyl acetate–toluene); [a]
+77.2° (c 1.04, CHCl3); 1H
D
NMR (500 MHz, CDCl3) d 7.95 (m, 2H, ArH), 7.89 (m, 2H, ArH),
7.49 (m, 2H, ArH), 7.34 (m, 7H, ArH), 7.20 (m, 2H, ArH), 5.78 (d,
1H, J1,2 5.2 Hz, H-1), 5.62 (dd, 1H, J1 ,2 7.9 Hz, J2 ,3 10.7 Hz, H-20),
0
0
0
0
00 00
5.54 (vt, 1H, J2,3 2.5 Hz, J3,4 2.5 Hz, H-3), 5.43 (dd, 1H, J3 ,4 3.1 Hz,
J2 ,3 11.1 Hz, H-300), 5.21 (dd, 1H, J3 ,4 3.1 Hz, J2 ,3 10.7 Hz, H-30),
00 00
0
0
0
0
C55H58O18: C, 63.36; H, 5.64. Found: C, 63.02; H, 5.73.
5.01 (d, 1H, J1 ,2 3.5 Hz, H-100), 4.72 (d, 1H, J1 ,2 7.8 Hz, H-10), 4.50
(m, 1H, H-500), 4.46 (m, 2H, H-6ab0), 4.38 (m, 2H, H-40, PhCH2O),
4.34 (ddd, 1H, J2,4 0.9 Hz, J2,3 3.1 Hz, J1,2 5.2 Hz, H-2), 4.18 (d, 1H, Jgem
00 00
0
0
3.1.10. 6-O-Acetyl-2,3-di-O-benzoyl-b-
(1?4)-3-O-acetyl-6-O-benzyl-1,2-O-[(S)-1-
(methoxycarbonyl)ethylidene]- -glucopyranoside (15)
D-galactopyranosyl-
0
00
00
a-
D
12.1 Hz, PhCH2O), 3.92 (m, 2H, H-4, H-5 ), 3.81 (m, 2H, H-2 , H-6a ),
3.75 (m, 4H, H-5, C(O)OCH3), 3.43 (m, 3H, H-6ab, H-6b00), 2.12 (s, 3H,
CH3C(O)O), 2.11 (s, 3H, CH3C(O)O), 2.07 (s, 3H, CH3C(O)O), 1.85 (s,
3H, CH3C(O)O), 1.70 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3) d
170.4 (C@O), 170.1 (C@O), 169.9 (C@O), 169.6 (C@O), 169.3
(C@O), 166.0 (C@O), 164.7 (C@O), 137.9 (Ar), 133.6 (Ar), 133.3
(Ar), 129.8 (Ar), 129.6 (Ar), 129.2 (Ar), 128.7 (Ar), 128.4 (Ar),
128.3 (Ar), 127.7 (Ar), 127.6 (Ar), 105.3 (quaternary C, pyruvate),
102.4 (C-10), 99.2 (C-100), 98.1 (C-1), 76.2 (C-50), 74.8 (C-40), 74.3
(C-2), 73.5 (C-30), 73.2 (PhCH2O), 72.1 (C-4), 70.7 (C-3), 69.5 (C-
Compound 14 (0.685 g, 0.737 mmol) was dissolved in anhy-
drous dichloromethane (24 mL) and placed under argon atmo-
sphere. The stirred solution was cooled to ꢁ20 °C in a dry ice/
acetone bath. TfOH (0.065 mL, 0.737 mmol) was added and the
reaction instantly turned purple indicating a complete reaction.
TLC (20%, ethyl acetate–toluene) confirmed the reaction to be com-
plete. The reaction mixture was neutralized with Et3N until neutral
and the contents were diluted with dichloromethane. This crude
mixture was washed with saturated aqueous sodium bicarbonate,
distilled water, and saturated aqueous sodium chloride. The organ-
ic phase was dried over anhydrous sodium sulfate, filtered, and
concentrated. The crude product was purified by flash column
chromatography on silica gel (2:1, hexanes–ethyl acetate) to give
the product 15 (0.483 g, 81%) as a white foam; Rf 0.22 (20%, ethyl
0
00
00
00
2 ), 68.6, 68.5 (C-5, C-3 ), 68.3 (C-6), 67.1, 67.0 (C-4 , C-5 ), 61.4
(C-60), 60.5 (C-600), 58.2 (C-200), 52.6 (CH3OC(O)), 21.3 (CH3C(O)O),
20.9 (CH3C(O)O), 20.8 (CH3C(O)O), 20.6 (CH3C(O)O), 20.5(5)
(CH3C(O)O), 20.5(1) (CH3C(O)O); ESI HRMS Calc0d. for C53H59N3O24-
Na 1144.3381. Found 1144.3376; Anal. Calcd for C53H59N3O24: C,
56.73; H, 5.30; N, 3.74. Found: C, 56.86; H, 5.42; N, 3.44.
acetate–toluene); [a]
+41.8° (c 1.08, CHCl3); 1H NMR (500 MHz,
D
CDCl3) d 7.99 (m, 2H, ArH), 7.93 (m, 2H, ArH), 7.54 (m, 2H, ArH),
7.37 (m, 7H, ArH), 7.22 (m, 2H, ArH), 5.80 (d, 1H, J1,2 5.1 Hz, H-1),
3.1.12. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-
galactopyranosyl-(1?4)-6-O-acetyl-2,3-di-O-benzoyl-b-
galactopyranosyl-(1?4)-3-O-acetyl-6-O-benzyl-1,2-O-[(S)-1-
(methoxycarbonyl)ethylidene]- -glucopyranoside (17)
a-D-
5.71 (dd, 1H, J1 ,2 7.9 Hz, J2 ,3 10.4 Hz, H-20), 5.57 (vt, 1H, J2,3
D-
0
0
0
0
0
0
0
0
2.8 Hz, J3,4 2.8 Hz, H-3), 5.24 (dd, 1H, J3 ,4 3.3 Hz, J2 ,3 10.4 Hz, H-
30), 4.70 (d, 1H, J1 ,2 7.9 Hz, H-10), 4.43 (dd, 1H, J5 ,6a 7.1 Hz, J6a ,6b
11.5 Hz, H-6a0), 4.40 (d, 1H, Jgem 12.2 Hz, PhCH2O), 4.36 (ddd, 1H,
a-D
0
0
0
0
0
0
Azide 16 (0.297 g, 0.265 mmol) was placed in a dry flask dis-
solved in a 6:1 mixture of acetonitrile:triethylamine (8 mL) and
placed under an argon atmosphere. Dithiothreitol (0.163 g,
1.06 mmol) was added and the reaction proceeded at room tem-
perature overnight. Subsequent TLC analysis (60%, ethyl acetate–
hexanes) showed no remaining starting material. A 1:1 mixture
of acetic anhydride:pyridine was added to the reaction flask and
again allowed to proceed overnight. The reaction mixture was then
concentrated to dryness while co-evaporating with toluene. The
crude product was then subjected to flash column chromatography
on silica gel (40%, acetone–hexanes) to provide the product 17
(0.262 g, 87%) as a white foam; Rf 0.30 (1:1, acetone–hexanes);
0
0
J2,4 1.2 Hz, J2,3 3.0 Hz, J1,2 5.2 Hz, H-2), 4.33 (dd, 1H, J5 ,6b 5.9 Hz,
J6a ,6b 11.4 Hz, H-6b0), 4.27 (m, 1H, J3 ,4 3.1 Hz, H-40), 4.18 (d, 1H,
Jgem 12.3 Hz, PhCH2O), 3.94 (m, 1H, H-4), 3.90 (m, 1H, H-50), 3.77
(s, 3H, C(O)OCH3), 3.73 (m, 1H, H-5), 3.43 (dd, 1H, J5,6a 2.4 Hz,
J6a,6b 10.9 Hz, H-6a), 3.40 (dd, 1H, J5,6b 3.5 Hz, J6a,6b 10.9 Hz, H-
0
0
0
0
6b), 2.45 (d, 1H, J4 ,OH 6.0 Hz, 40-OH), 2.12 (s, 3H, CH3C(O)O), 2.11
0
(s, 3H, CH3C(O)O), 1.71 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3) d
170.9 (C@O), 169.5 (C@O), 169.3 (C@O), 165.8 (C@O), 164.9
(C@O), 137.9 (Ar), 133.5 (Ar), 133.3 (Ar), 129.9 (Ar), 129.7 (Ar),
129.3 (Ar), 129.0 (Ar), 128.5 (Ar), 128.4 (Ar), 127.8 (Ar), 105.4 (C,
pyruvate), 102.6 (C-10), 98.2 (C-1), 76.1 (C-4), 74.0 (C-2, C-30),
73.2 (PhCH2O), 72.2 (C-50), 70.5 (C-3), 69.5 (C-20), 68.7 (C-5), 68.3
(C-6), 67.1 (C-40), 61.9 (C-60), 52.6 (C(O)OCH3), 21.2 (CH3C(O)O),
20.9 (CH3C(O)O), 20.8 (CH3, pyruvate); ESI HRMS Calcd for
[a]
+77.2° (c 1.12, CHCl3); 1H NMR (500 MHz, CDCl3) d 7.94 (m,
D
2H, ArH), 7.91 (m, 2H, ArH), 7.51 (m, 2H, ArH), 7.41–7.31 (m, 7H,
00
ArH), 7.21 (m, 2H, ArH), 6.05 (d, 1H, J2 ,NH 9.5 Hz, N–H), 5.80 (d,
1H, J1,2 5.1 Hz, H-1), 5.69 (dd, 1H, J1 ,2 7.9 Hz, J2 ,3 10.5 Hz, H-20),
0
0
0
0
C
C
41H44O17Na 831.2471. Found 831.2473; Anal. Calcd for
41H44O17: C, 60.89; H, 5.48; O, 33.63. Found: C, 61.06; H, 5.54.
5.61 (m, 1H, H-3), 5.35 (m, 1H, H-400), 5.29 (dd, 1H, J3 ,4 3.1 Hz,
00 00
J2 ,3 11.5 Hz, H-300), 5.24 (dd, 1H, J3 ,4 3.0 Hz, J2 ,3 10.6 Hz, H-30),
00 00
0
0
0
0
5.06 (d, 1H, J1 ,2 3.9 Hz, H-100), 4.71 (d, 1H, J1 ,2 7.8 Hz, H-10), 4.69
00 00
0
0
3.1.11. 3,4,6-tri-O-Acetyl-2-azido-2-deoxy-
galactopyranosyl-(1?4)-6-O-acetyl-2,3-di-O-benzoyl-b-
galactopyranosyl-(1?4)-3-O-acetyl-6-O-benzyl-1,2-O-[(S)-1-
(methoxycarbonyl)ethylidene]- -glucopyranoside (16)
a-
D
-
(ddd, 1H, J1 ,2 3.9 Hz, J2 ,NH 9.7 Hz, J2 ,3 11.6 Hz, H-200), 4.47 (dd,
00 00
00
00 00
D-
1H, J5 ,6a 6.2 Hz, J6a ,6b 11.2 Hz, H-6a0), 4.43 (d, 1H, J3 ,4 3.0 Hz, H-
0
0
0
0
0
0
40), 4.39 (m, 2H, Jgem 12.2 Hz, H-5 , PhCH2O), 4.33 (ddd, 1H, J2,4
00
a-
D
The acceptor 15 (0.203 g, 0.251 mmol) and 4 Å MS (100 mg)
were combined in a dry flask and dissolved in anhydrous diethyl
ether (2 mL). The reaction mixture was stirred for 1 h under argon
atmosphere, after which the activator TMSOTf (6.8 mL,
1.0 Hz, J2,3 2.5 Hz, J1,2 5.1 Hz, H-2), 4.19 (d, 1H, Jgem 12.1 Hz,
PhCH2O), 4.02 (dd, 1H, J5 ,6b 8.2 Hz, J6a ,6b 11.2 Hz, H-6b0), 3.94
0
0
0
0
(m, 2H, H-4, H-50), 3.77 (s, 3H, C(O)OCH3), 3.74 (m, 1H, H-5), 3.68
(dd, 1H, J5 ,6a 8.5 Hz, J6a ,6b 10.9 Hz, H-6a00), 3.46 (dd, 1H, J5,6a
2.2 Hz, J6a,6b 10.9 Hz, H-6a), 3.40 (dd, 1H, J5,6b 3.5 Hz, J6a,6b
00
00
00
00
0.038 mmol) was added. The trichloroacetimidate donor
5
10.8 Hz, H-6b), 3.20 (dd, 1H, J5 ,6b 6.1 Hz, J6a ,6b 10.9 Hz, H-6b00),
2.14 (s, 3H, CH3C(O)O), 2.12 (s, 3H, CH3C(O)O), 2.09 (s, 3H,
CH3C(O)O), 2.08 (s, 3H, CH3C(O)O), 2.04 (s, 3H, CH3C(O)O), 1.77
(s, 3H, CH3C(O)NH), 1.73 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3)
00
00
00
00
(0.478 g, 1.00 mmol) was dissolved in anhydrous diethyl ether
(2 mL) and added dropwise at room temperature to the stirred solu-
tion. The reaction was allowed to proceed for 2 h and reaction pro-
gress was monitored by TLC analysis (60%, ethyl acetate–hexanes)

12
J. M. Jacobson et al. / Carbohydrate Research 378 (2013) 4–14
d 170.7 (C@O), 170.2 (C@O), 169.7 (C@O), 169.4 (C@O), 169.2
(C@O), 165.8 (C@O), 164.9 (C@O), 137.9 (Ar), 133.6 (Ar), 133.4
(Ar), 129.7 (Ar), 129.6 (Ar), 129.1 (Ar), 128.8 (Ar), 128.6 (Ar),
128.5 (Ar), 128.4 (Ar), 127.8 (Ar), 127.7 (Ar), 105.5 (quaternary C,
pyruvate), 102.6 (C-10), 98.2 (C-100), 97.9 (C-1), 76.4 (C-4), 74.1
(C-2), 73.2 (PhCH2O), 73.1 (C-30), 72.2 (C-40), 72.1 (C-50), 70.0 (C-
3), 69.6 (C-20), 68.3 (C-5), 68.2 (C-6), 68.1 (C-300), 66.8, 66.7 C-400,
-500), 60.6, 60.5 (C-60, C-600), 52.7 (CH3OC(O)), 47.6 (C-200), 23.2
(CH3C(O)O), 21.2 (CH3C(O)O), 20.9 (CH3C(O)O), 20.8 (CH3C(O)O),
20.7 (CH3, pyruvate), 20.5 (CH3C(O)NH); ESI HRMS calcd for
that no starting material remained. The reaction was diluted with
dichloromethane and washed with saturated aqueous sodium
bicarbonate, distilled water, and saturated aqueous sodium chlo-
ride. The organic layer was dried over anhydrous sodium sulfate, fil-
tered, and concentrated to dryness. The crude product was purified
by flash column chromatography on silica gel (1:1, acetone–hex-
anes) to give the product 18 (0.165 g, 99%) was a white foam; Rf
0.26 (1:1, acetone–hexanes); [a]
+92.2° (c 1.08, CHCl3); 1H NMR
D
(600 MHz, CDCl3) d 8.27 (m, 2H, ArH), 7.93 (m, 4H, ArH), 7.48 (m,
2H, ArH), 7.38 (m, 2H, ArH), 7.32 (m, 2H, ArH), 7.28 (m, 2H, ArH),
00
C
C
55H63NO25Na 1160.3581. Found 1160.3573; Anal. Calcd for
55H63NO25: C, 58.04; H, 5.58; N, 1.23. Found: C, 57.76; H, 5.57;
6.02 (d, 1H, J2 ,NH 9.5 Hz, N–H), 5.79 (d, 1H, J1,2 5.2 Hz, H-1), 5.72
(dd, 1H, J1 ,2 7.7 Hz, J2 ,3 10.5 Hz, H-20), 5.64 (m, 1H, H-3), 5.34 (m,
0
0
0
0
1H, H-400), 5.32 (dd, 1H, J3 ,4 3.1 Hz, J2 ,3 10.6 Hz, H-30), 5.29 (dd,
0
0
0
0
N, 1.28.
1H, J3 ,4 3.2 Hz, J2 ,3 11.6 Hz, H-300), 5.06 (d, 1H, J1 ,2 3.9 Hz, H-
00 00
00 00
00 00
100), 4.94 (d, 1H, J1 ,2 7.8 Hz, H-10), 4.66 (ddd, 1H, J1 ,2 3.8 Hz, J2 ,NH
0
0
00 00
00
3.1.13. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-
galactopyranosyl-(1?4)-6-O-acetyl-2,3-di-O-benzoyl-b-
galactopyranosyl-(1?4)-3-O-acetyl-1,2-O-[(S)-1-
(methoxycarbonyl)ethylidene]-a-D-glucopyranoside (3)
a-
D-
9.6 Hz, J2 ,3 11.5 Hz, H-200), 4.50 (m, 1H, H-6a0), 4.44 (d, 1H, J3 ,4
00 00
0
0
D-
3.1 Hz, H-40), 4.37 (m, 2H, H-2, H-500), 4.32 (dd, 1H, J5,6a 2.2 Hz,
J6a,6b 11.7 Hz, H-6a), 4.16 (dd, 1H, J5,6b 4.9 Hz, J6a,6b 11.8 Hz, H-6b),
4.00 (m, 3H, J5,6a 2.2 Hz, J5,6b 4.9 Hz, J4,5 9.5 Hz, H-5, H-50, H-6b0),
3.83 (m, 1H, J4,5 9.5 Hz, H-4), 3.78 (s, 3H, C(O)OCH3), 3.67 (m, 1H,
The 6-O-benzyl trisaccharide derivative 17 (0.738 g,
0.648 mmol) was combined with 20% wt. Pd(OH)2 on charcoal
(0.250 g) in the reaction flask and the starting material was dis-
solved in ethyl acetate (14 mL). The reaction vessel was placed un-
der a hydrogen atmosphere and the hydrogenation reaction was
allowed to proceed at room temperature for 3 h. TLC analysis
(1:1, acetone–hexanes) showed the reaction to be complete. The
reaction mixture was filtered through a Millipore filter to remove
the solid catalyst and the filtrate was subsequently concentrated
to dryness to provide the product 3 (0.660 g, 97%) as a white foam;
J5 ,6a 8.4 Hz, J6a ,6b 10.9 Hz, H-6a00), 3.21 (dd, 1H, J5 ,6b 6.1 Hz,
00
00
00
00
00
00
J6a ,6b 11.0 Hz, H-6b00), 2.13 (s, 3H, CH3C(O)O), 2.12 (s, 3H,
CH3C(O)O), 2.08 (s, 3H, CH3C(O)O), 2.07 (s, 3H, CH3C(O)O), 2.03 (s,
3H, CH3C(O)O), 1.78 (s, 3H, CH3C(O)NH), 1.76 (s, 3H, CH3); 13C
NMR (125 MHz, CDCl3) d 170.7 (C@O), 170.6 (C@O), 170.2 (C@O),
169.7 (C@O), 169.2 (C@O), 165.8 (C@O), 165.0 (Ar), 155.3 (Ar),
151.8 (Ar), 145.4 (Ar), 133.7 (Ar), 133.5 (Ar), 129.7 (Ar), 129.6
(Ar), 128.8 (Ar), 128.7 (Ar), 128.5 (Ar), 128.4 (Ar), 105.7 (quaternary
C, pyruvate), 102.5 (C-10), 98.3 (C-100), 97.7 (C-1), 77.0 (C-4), 74.2 (C-
2), 73.1 (C-30), 72.3, 72.2 (C-40, C-50), 69.6, 69.5(9) (C-3, C-20), 68.0
(C-300), 67.4 (C-5), 66.8 (C-6), 66.6, 66.5 (C-400, C-500), 60.6 (C-600),
60.5 (C-60), 52.7 (CH3OC(O)), 47.7 (C-200), 23.1 (CH3C(O)O), 21.2
(CH3C(O)O), 20.9 (CH3C(O)O), 20.7(8) (CH3C(O)O), 20.7(6)
(CH3C(O)O), 20.6 (CH3C(O)NH), 20.5 (CH3, pyruvate); ESI HRMS
calcd for C55H60N2O29Na 1235.3174. Found 1235.3167; Anal. Calcd
for C55H60N2O29: C, 54.46; H, 4.99; N, 2.31. Found: C, 54.45; H, 5.22;
N, 2.19.
00
00
Rf 0.24 (1:1, acetone–hexanes); [a]
D +81.6° (c 1.11, CHCl3); 1H NMR
(500 MHz, CDCl3) d 7.93 (m, 2H, ArH), 7.90 (m, 2H, ArH), 7.48 (m,
00
2H, ArH), 7.36 (m, 4H, ArH), 6.28 (d, 1H, J2 ,NH 10.4 Hz, N–H),
5.74 (m, 2H, H-1, H-20), 5.50 (m, 1H, H-3), 5.33 (m, 1H, H-400),
5.30 (dd, 1H, J3 ,4 3.2 Hz, J2 ,3 11.4 Hz, H-300), 5.24 (dd, 1H, J3 ,4
00 00
00 00
0
0
2.9 Hz, J2 ,3 10.5 Hz, H-30), 5.01 (d, 1H, J1 ,2 3.8 Hz, H-100), 4.93 (d,
0
0
00 00
1H, J1 ,2 8.0 Hz, H-10), 4.65 (ddd, 1H, J1 ,2 3.8 Hz, J2 ,NH 9.6 Hz,
0
0
00 00
00
J2 ,3 11.4 Hz, H-200), 4.46 (dd, 1H, J5 ,6a 4.2 Hz, J6a ,6b 9.0 Hz, H-
00 00
0
0
0
0
6a0), 4.42 (d, 1H, J3 ,4 2.9 Hz, H-40), 4.38 (m, 1H, H-500), 4.32 (ddd,
1H, J2,4 1.0 Hz, J2,3 2.6 Hz, J1,2 5.1 Hz, H-2), 4.00 (m, 3H, H-4, H-50,
H-6b0), 3.74 (s, 3H, C(O)OCH3), 3.66 (m, 3H, H-5, H-6a, H-6a00),
0
0
3.1.15. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-
galactopyranosyl-(1?4)-6-O-acetyl-2,3-di-O-benzoyl-b-D-
a-D-
00
00
00
00
3.49 (m, 1H, H-6b), 3.10 (dd, 1H, J5 ,6b 6.0 Hz, J6a ,6b 10.9 Hz, H-
6b00), 2.10 (s, 3H, CH3C(O)O), 2.09 (s, 3H, CH3C(O)O), 2.06 (s, 3H,
CH3C(O)O), 2.05 (s, 3H, CH3C(O)O), 2.01 (s, 3H, CH3C(O)O), 1.76
(s, 3H, CH3C(O)NH), 1.72 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3)
d 170.8 (C@O), 170.7 (C@O), 170.2 (C@O), 169.7 (C@O), 169.3
(C@O), 169.2 (C@O), 165.9 (C@O), 165.1 (C@O), 133.7 (Ar), 133.5
(Ar), 129.7 (Ar), 129.6 (Ar), 128.9 (Ar), 128.7 (Ar), 128.5 (Ar),
105.6 (quaternary C, pyruvate), 102.3 (C-10), 98.6 (C-100), 97.8 (C-
1), 75.3 (C-4), 73.9 (C-2), 73.5 (C-30), 72.6 (C-40), 72.3 (C-50), 70.2
(C-3), 69.2, 68.9 (C-5, C-20), 67.9 (C-300), 66.8, 66.7 (C-400, C-500),
61.5 (C-600), 60.6, 60.4 (C-6, C-60), 52.7 (C(O)OCH3), 47.7 (C-200),
23.1 (CH3C(O)O), 21.2 (CH3C(O)O), 20.9 (CH3C(O)O), 20.8
(CH3C(O)O), 20.6 (CH3C(O)NH), 20.5 (CH3–pyruvate); ESI HRMS
calcd for C48H57NO25Na 1070.3112. Found 1070.3105; Anal. Calcd
for C48H57NO25: C, 55.01; H, 5.48; N, 1.34. Found: C, 55.04; H,
5.65; N, 1.38.
galactopyranosyl-(1?4)-3-O-acetyl-6-O-(10-oxo-3,6,11-trioxa-
9-azatetradec-13-ynyl)carbamoyl-1,2-O-[(S)-1-
(methoxycarbonyl)ethylidene]-
The p-nitrophenylcarbamoyl
0.541 mmol) was combined with the amine
0.811 mmol) and dissolved in dry CH2Cl2 (10 mL). Et3N
(0.151 mL, 1.08 mmol) was added and the reaction proceeded at
room temperature for 2 h. The reaction mixture was concentrated
to dryness and the crude product purified by flash column chroma-
tography on silica gel (1:1, acetone–hexanes) to provide the prod-
uct 19 (0.706 g, 95%) as a white foam; Rf 0.34 (1:1, acetone–
a-D-glucopyranoside (19)
derivative
18
(0.656 g,
4
(0.186 g,
hexanes); [a]
+70.7° (c 1.00, CHCl3); 1H NMR (500 MHz, CDCl3) d
D
7.95 (m, 2H, ArH), 7.92 (m, 2H, ArH), 7.49 (m, 2H, ArH), 7.37 (m,
00
4H, ArH), 6.07 (d, 1H, J2 ,NH 9.3 Hz, N–H), 5.75 (d, 1H, J1,2 5.2 Hz,
H-1), 5.70 (dd, 1H, J1 ,2 7.8 Hz, J2 ,3 10.5 Hz, H-20), 5.64 (br s, 1H,
0
0
0
0
H-3), 5.40 (br s, 1H, N–H linker), 5.34 (m, 1H, J3 ,4 2.8 Hz, H-400),
00 00
00 00
3.1.14. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-
a-
D-
5.29 (t, 1H, J3 ,4 2.8 Hz, H-300), 5.26 (m, 1H, H-30), 5.04 (d, 1H,
J1 ,2 3.9 Hz, H-100), 4.89 (d, 1H, J1 ,2 7.8 Hz, H-10), 4.66 (m, 3H,
00 00
0
0
galactopyranosyl-(1?4)-6-O-acetyl-2,3-di-O-benzoyl-b-
galactopyranosyl-(1?4)-3-O-acetyl-6-(4-
nitrophenylcarbonate)-1,2-O-[(S)-1-
D-
00 00
00
0
0
0
0
J1 ,2 3.8 Hz, H-2 , OCH2C„CH), 4.48 (dd, 1H, J5 ,6a 9.7 Hz, J6a ,6b
14.3 Hz, H-6a0), 4.43 (d, 1H, J3 ,4 3.1 Hz, H-40), 4.37 (m, 1H, H-500),
4.31 (dd, 1H, J2,3 2.5 Hz, J1,2 5.2 Hz, H-2), 4.18 (dd, 1H, J5,6a 4.2 Hz,
J6a,6b 11.8 Hz, H-6a), 4.03 (dd, 1H, J5,6b 1.2 Hz, J6a,6b 11.6 Hz, H-
6b), 3.98 (m, 2H, H-50, H-6b0), 3.82 (m, 1H, H-5), 3.75 (s, 3H,
0
0
(methoxycarbonyl)ethylidene]-a-D-glucopyranoside (18)
The primary alcohol 3 (0.144 g, 0.137 mmol) was combined with
4-nitrophenyl chloroformate (0.033 g, 0.165 mmol) and dissolved
in dry dichloromethane (3 mL). Pyridine (0.022 mL, 0.274 mmol)
was then added and the reaction allowed to proceed at room tem-
perature for 10 min. TLC analysis (1:1, acetone–hexanes) showed
00
00
00
00
C(O)OCH3), 3.72 (m, 1H, H-4), 3.66 (dd, 1H, J5 ,6a 8.5 Hz, J6a ,6b
10.8 Hz, H-6a00), 3.60 (s, 4H, OCH2CH2O linker), 3.55 (m, 4H,
HNCH2CH2O linker), 3.36 (m, 4H, HNCH2CH2O), 3.15 (dd, 1H,

J. M. Jacobson et al. / Carbohydrate Research 378 (2013) 4–14
13
J5 ,6b 6.0 Hz, J6a ,6b 10.8 Hz, H-6b00), 2.48 (t, 1H, JCH,CH2 2.1 Hz,
OCH2C„CH), 2.10 (s, 3H, CH3C(O)O), 2.09 (s, 3H, CH3C(O)O), 2.07
(s, 3H, CH3C(O)O), 2.06 (s, 3H, CH3C(O)O), 2.02 (s, 3H, CH3C(O)O),
1.75 (s, 3H, CH3 pyruvate), 1.72 (s, 3H, CH3C(O)NH); 13C NMR
(125 MHz, CDCl3) d 170.7 (C@O), 170.6 (C@O), 170.2 (C@O),
170.1 (C@O), 169.7 (C@O), 169.3 (C@O), 168.9 (C@O), 165.8
(C@O), 165.1 (C@O), 155.8 (C@O), 155.5 (C@O), 133.6 (Ar), 133.3
(Ar), 129.7 (Ar), 129.7 (Ar), 129.6 (Ar), 128.9 (Ar), 128.7 (Ar),
128.5 (Ar), 128.4 (Ar), 105.6 (quaternary C, pyruvate), 102.3 (C-
10), 98.4 (C-100), 97.7 (C-1), 76.4 (C-4), 74.7 (OCH2C„CH), 73.9 (C-
2), 73.3 (C-300), 72.3, 72.1 (C-40, C-50), 70.3, (CH2, linker), 70.0
(CH2, linker), 69.9 CH2 linker), 69.8 (C-3), 69.5 (C-20), 68.0 (C-30),
67.5 (C-5), 66.8, 66.7 (C-400, C-500), 63.4 (C-6), 60.4 (C-6, C-60), 52.7
(C(O)OCH3), 52.4 (OCH2C„CH), 47.6 (C-200), 40.9 (CH2 linker),
23.1 (CH3C(O)O), 21.1 (CH3C(O)O), 20.9 (CH3C(O)O), 20.8
(CH3C(O)O), 20.6 (CH3C(O)NH), 20.4 (CH3, pyruvate); ESI HRMS
calcd for C59H73N3O30Na 1326.4171. Found 1326.4159; Anal. Calcd
for C59H70N3O30: C, 54.33; H, 5.64; N, 3.22. Found: C, 54.43; H,
5.79; N, 3.31.
3.1.17. PolyBAIT-PkNAc (1)
00
00
00
00
The heterobivalent monomer 2 (0.113 g, 0.130 mmol) was com-
bined with poly[acrylamide-co-(2-azidopropylmethacrylamide)]
(27 kDa, PDI 1.29,27 0.139 g, and 0.0865 mmol) and the contents
dissolved in degassed MilliQ water (0.5 mL). A 1.0 M solution of so-
dium ascorbate was prepared and added to the solution (125
followed by the addition of a 0.005 M solution of CuSO4 (250
l
L)
l
L).
The reaction proceeded for 2 days with stirring and subsequent
TLC (4:5:1:0.1, dichloromethane:methanol:water:acetic acid)
showed the reaction to be incomplete. The pH of the reaction
was checked and found to be neutral. Sodium bicarbonate was
added until the pH was ꢃ8 and the reaction was allowed to con-
tinue for 24 h at which point TLC analysis showed the reaction to
be complete. The reaction product was isolated and purified via
dialysis and the pure product lyophilized from water to give the
PolyBAIT-PkNAc 1 product as a white solid; Characterization of
PolyBAIT-PkNAc required comparison of the spectrum of starting
monomer with that obtained for conjugated polymer (Supplemen-
tary data). The percent incorporation of monomer to polymer, was
established by integrating the resonance of an anomeric proton
with resonances of the polymer backbone. Specifically, the integra-
tion of peak A (d 5.61, d, 1H, and H-1 ligand) was compared to res-
onances labeled peak B (d 2.35–1.28, br m, 67H, –CH2–, –CH–
polymer backbone, –CH3 pyruvate, and –CH3 NHAc). Since this
group included six protons from ligand pyruvate and acetamido
groups, 6H were subtracted from the total of peak B, leaving
61H. As each acrylamide monomer has 3H, we have a total of
approximately 20 acrylamide residues to 1 carbohydrate residue.
This corresponds to a 5% incorporation of the carbohydrate ligand
to the polymer.
3.1.16. 2-Acetamido-2-deoxy-a-D-galactopyranosyl-(1?4)-b-D-
galactopyranosyl-(1?4)-6-O-(10-oxo-3,6,11-trioxa-9-
azatetradec-13-ynyl)carbamoyl-1,2-O-[(S)-1-
(carboxy)ethylidene]-a-D-glucopyranoside (2)
The acetylated trisaccharide 19 (0.410 g, 0.314 mmol) was
placed in a round bottomed flask and dissolved in anhydrous
methanol (20 mL). A 1 M solution of CH3ONa in methanol was
prepared and added (0.314 mL, 0.314 mmol) to the reaction flask.
The reaction was allowed to proceed for 2 days at which point TLC
(10%, methanol–dichloromethane) showed the reaction was com-
plete. The reaction mixture was concentrated to dryness and
placed under high vacuum for 2 h. The residue was subsequently
dissolved in 20 mL MilliQ water and the pH checked and adjusted
to ensure a pH ꢃ8/9. The hydrolysis of the methyl ester was al-
lowed to proceed for 15 min at which point TLC analysis
Acknowledgements
This research was funded by the Alberta Glycomics Centre and
NSERC. JMJ was the recipient of NSERC CGS-D scholarship.
(4:5:1:0.1,
dichloromethane–methanol–water–acetic
acid)
showed no remaining starting material. The reaction was
quenched by dropwise addition of TFA until the pH was ꢃ7. The
reaction mixture was concentrated and lyophilized. The crude
product was purified by HPLC on an X-Bridge C18 reverse-phase
column (100% water (0.1%, TFA) ? 100% acetonitrile) and was
once again lyophilized to provide the product 2 (0.242 g, 88%)
was a white powder; Rf 0.58 (4:5:1:0.1, dichloromethane–metha-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2013.
05.010.
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[
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00 00
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