Synthesis of modified Trichinella spiralis disaccharide epitopes and a comparison of their recognition by chemical mapping and saturation transfer difference NMR

Article abstract of DOI:10.1016/j.carres.2013.10.012

A rat monoclonal antibody 9D4 raised against the cell surface N-glycan of the parasite Trichinella spirallis protects rats against further infection. The terminal disaccharide β-d-Tyvp(1→3)β-d-GalNAcp (2) represents the immunodominant portion of the antig

Full text of DOI:10.1016/j.carres.2013.10.012

Carbohydrate Research 383 (2014) 1–13
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of modified Trichinella spiralis disaccharide epitopes
and a comparison of their recognition by chemical mapping and
saturation transfer difference NMR
b
c
c
Lina Cui a, , Chang-Chun Ling , Joanna Sadowska , David R. Bundle
⇑
a Department of Radiology, Molecular Imaging Program at Stanford and Bio-X Program, School of Medicine, Stanford University, CA 94305, United States
b Department of Chemistry, Alberta Glycomics Centre, University of Calgary, Calgary, Alberta T2N 1N4, Canada
c Department of Chemistry, Gunning-Lemieux Chemistry Centre, Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
A rat monoclonal antibody 9D4 raised against the cell surface N-glycan of the parasite Trichinella spirallis
Received 10 September 2013
Received in revised form 22 October 2013
Accepted 23 October 2013
protects rats against further infection. The terminal disaccharide b-D-Tyvp(1?3)b-D-GalNAcp (2) repre-
sents the immunodominant portion of the antigenic determinant. Chemical mapping of the antibody
binding site by functional group modification employing monodeoxy and mono-O-methyl congeners
identified key polar contacts and topography of the bound disaccharide. We report here a comparison
of the chemical mapping studies with the antigen topography inferred from saturation transfer difference
(STD) NMR experiments. During chemical mapping several congeners of compound 2 showed substan-
tially enhanced binding. Pairing of these functional group modifications to create derivatives 6 and 7
did not show additive free energy gains and STD NMR data point to small variations in mode of binding
as a probable cause. Improved syntheses of disaccharides 2–7 are reported.
Available online 1 November 2013
Keywords:
Protein carbohydrate interaction
Trichinella spirallis
Synthesis of cell surface N-glycan
Tyvelose
Ó 2013 Elsevier Ltd. All rights reserved.
1,2-cis-b Glycosidic linkages
Saturation transfer difference (STD) NMR
1. Introduction
derivative 3 is a sixfold better binder than the native disaccharide
2. The hydroxyl group at C-6 is clearly at the periphery of the bind-
The monoclonal antibody (mAb) IgG 9D4 raised against
Trichinella spiralis, a pathogenic nematode that causes trichinosis,
exhibits high affinity for the terminal tetrasaccharide 1 of the cell
surface N-glycan of the pathogen (Fig. 1).1,2 The binding site of
the antibody contacts all four sugar moieties, while the majority
of its binding energy is focused around the rare dideoxyhexose
ing site, as deoxygenation has little impact on the binding, while O-
methylation (compound 4) provides an 18-fold affinity gain, most
likely by the introduction of new van der Waals’ interactions on the
edge of the binding pocket. A similar situation occurs for the 2-O-
methyl derivative of the tyvelose residue (disaccharide 5), with a
ninefold activity gain.
Saturation transfer difference (STD) NMR reveals the relative
proximity of each proton of the ligand to the protein, thus provid-
ing a complementary approach to epitope mapping.6 In this paper,
STD NMR experiments were carried out to analyze the interaction
between mAb 9D4 and a series of disaccharide ligands 2–7, for
which a concise and improved synthetic strategy was developed.
The results from STD NMR studies are compared with the previous
chemical mapping studies.5 In addition, paired functional group
modifications in disaccharides 6 and 7 (Fig. 1) were investigated
by competitive inhibition and STD NMR.
containing disaccharide b-
D
-Tyvp-(1?3)-b-
D
-GalpNAc 2.2–4 The
recognition surface of 2 that is accepted in the IgG 9D4 antibody
binding site was identified by a chemical mapping study.5 The
activity changes accompanying monodeoxy and mono-O-methyla-
tion of each hydroxyl group of 2 established that polar contacts in-
volved the tyvelose and GalNAc C-4 hydroxyl groups, with tyvelose
C2 and GalNAc C4 and C6 hydroxyl group sitting close to or at the
periphery of the binding site. The acetamido group of the GalNAc
moiety is not crucial to binding, as its replacement by a hydroxyl
group has only a subtle effect on activity. It should also be noted
that a disaccharide in which a GlcNAc residue replaces GalNAc is
inactive indicating steric constraints at the C4 position of GalNAc.
During the course of functional group modification several sub-
stantial gains in affinity were observed. The free amino sugar
2. Results and discussion
2.1. Synthesis of disaccharides 2–7
To get easy access to a library of ligands containing the rare
sugar tyvelose and a challenging 1,2-cis b glycosidic linkage, an
⇑
Corresponding author. Tel.: +1 650 736 2456.
E-mail address: linacui@stanford.edu (L. Cui).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.10.012

2
L. Cui et al. / Carbohydrate Research 383 (2014) 1–13
O
O
HO
OH
O
O
N
HO
HO
a
OH
O
NHAc
15
16
OR
O
OR
O
HO
HO
HO
PivO
b
d
c
OMe
OMe
NHAc
NHAc
17 R = H
18 R = Piv
e
20 R = Me
21 R = Me
PivO
HO
OR
O
OMe
NHAc
19 R = Piv
22 R = Me
Figure 1. Structure of native terminal tetrasaccharide of the N-glycan expressed on
the cell surface of Trichinella spiralis parasite, and its disaccharide congener 2.
Synthetic disaccharide targets, native terminal disaccharide 2, disaccharides (3, 4,
and 5) with higher affinity to IgG 9D4 than 2 based on the previous chemical mapping
study, and disaccharides with paired functional group modification (6 and 7).
Scheme 2. Synthesis of acceptors. Reaction conditions: (a) FeCl3, acetone, reflux,
20 min; (b) MeOH, p-TsOH, rt, 81% over two steps; (c) PivCl, pyridine, 90%; (d) Tf2O,
pyridine, then H2O, reflux, 89%; (e) (Bu3Sn)2O, toluene, reflux; then MeI, 80% over
two steps.
efficient and concise synthetic strategy is desirable. The target
disaccharides 2–7 were synthesized using the new approach that
started from acetonides of glucofuranose and 2-acetamido-2-
deoxy glucofuranose (Schemes 1–3).
Synthesis of the tyvelose moiety was envisaged as proceeding
via a 3-deoxy glucose donor with manipulation of stereochemistry
at C2 and functionality at C6 conducted on disaccharide deriva-
tives. Barton–McCombie deoxygenation7 of 1,2:5,6-di-O-isopropyl-
afford 13. The glycosyl donor 14 was obtained following acetyla-
tion of 13. Compounds 11 to 14 could be isolated to obtain
b separately; however, a mixture of /b isomers ( /b = 1/2) of the
donor 14 was employed for the glycosylation.11
Previously, the selectively protected GalNAc acceptor was syn-
a and
a
a
thesized from 2-acetamido-2-deoxy-D-galactose by formation of a
4,6-O-benzylidene acetal.5 However, further modifications of the
disaccharides were low yielding. A facile synthesis of 2-acetam-
idene-
Hydrolysis of the isopropylidene protecting groups and acetylation
gave an anomeric mixture (
/b = 1/2) of pyranose 10,8 This tetra-
a-D-glucofuranose 8 gave 3-deoxy furanose 9 (Scheme 1).
ido-2-deoxy-b-
D
D-glucopyranosides from 2-acetamido-2-deoxy-
-glucose via a furanosyl oxazoline was reported by our group,12
a
and here we adopted the methodology to provide an inexpensive
and convenient route to GalNAc acceptors. Briefly, 15 was stirred
with anhydrous ferric chloride in acetone at reflux to give oxazoline
16,13 and methyl pyranoside 17 was generated after treatment with
a stoichiometric amount of p-toluenesulfonic acid in methanol
(Scheme 2). The 3- and 6-hydroxyl groups of compound 17 were
protected selectively as pivaloate esters14 to afford 18 (Scheme 2).
Treatment with triflic anhydride provided a 4-O-triflate, which re-
acted in situ with water to achieve a configurational change from
glucose to galactose and a convenient migration of pivaloyl group
from O-3 to O-4.15,16
acetate was converted to thiophenyl glycoside 11 by reaction with
boron trifluoride and thiophenol.9,10 Transesterification gave 12
and subsequently 4,6-O-benzylidene acetal was installed to
O
O
O
HO
O
O
O
O
O
c, d
a, b
O
O
8
9
In order to increase the reactivity of the acceptor and to limit
modifications performed on disaccharides, methylation of the 6-hy-
droxyl group of the GalNAc moiety was performed on compound 17.
Treatment of 17 with bistributyltin oxide17 under azeotropic condi-
tions generated a tributylstannyl ether, which was subsequently
displaced by methyl iodide to obtain the desired methylation prod-
uct 20 (Scheme 2). The reactions to transform compound 20 to 22
resembled those employed for the transformation of compound
17 to 19.
Thiolglycoside 14 was activated by N-iodosuccinimide/triflic
acid,9,10 and glycosylation of 19 and 22 gave disaccharides 23
and 24 in good yield (Scheme 3). Selective removal of the 20-O-
acetyl group in the presence of the pivaloyl group was achieved
by stirring compounds 23 and 24 in a methanolic solution of gua-
nidine and sodium methoxide to generate disaccharides 25 and
26.18
OAc
O
OAc
O
e
f
AcO
Ph
AcO
OAc
SPh
OAc
11
OAc
10
OH
O
O
O
g
O
HO
SPh
O
SPh
OH
12
Ph
OH
13
O
h
O
SPh
OAc
Disaccharides 25 and 26 were each oxidized by Swern-type oxi-
dation,19,20 followed by reduction of the respective ketones using
14
L
-selectride21 in THF to give the epimers 27 and 29. Methylation
Scheme 1. Synthesis of donor. Reaction conditions: (a) NaH, imidazole, then CS2,
then MeI; (b) Bu3SnH, AIBN, 75% over two steps; (c) 60% HOAc–H2O, 80 °C; (d) Ac2O,
pyridine, 93% over two steps; (e) PhSH, BF3ꢀOEt2, 79%; (f) NaOMe, MeOH,
quantitative; (g) PhCH(OMe)2, CSA, 96%; (h) Ac2O, pyridine, 98%.
of 27 by treatment of MeI/NaH gave the methylated tyvelose deriv-
ative 28. A Hanessian–Hullar reaction converted compounds 27,
28, and 29 to the corresponding 6-bromo-6-deoxy disaccharides

L. Cui et al. / Carbohydrate Research 383 (2014) 1–13
3
PivO
PivO
HO
Ph
Ph
O
Ph
O
OR
O
OR
O
O
a
O
b
O
O
+
OMe
OMe
O
SPh
OAc
NHAc
OAc
NHAc
14
19 R = Piv
22 R = Me
23 R = Piv (85%)
24 R = Me (75%)
PivO
PivO
PivO
O
O
OR
O
Ph
O
OR
O
Br
BzO
OR
O
OR'
O
OR'
c, d
O
f
O
O
O
OMe
O
OMe
OMe
O
NHAc
NHAc
NHAc
OH
30 R = Piv, R' = H (82%)
31 R = Piv, R' = Me (72%)
32 R = Me, R' = H (82%)
27 R = Piv, R' = H (76%)
e
25 R = Piv (85%)
26 R = Me (95%)
28 R = Piv, R' = Me
29 R = Me, R' = H (86%)
PivO
HO
OR
O
OR
O
OR'
O
OR'
g
h
O
BzO
HO
OMe
OMe
O
O
NHAc
NHR"
33 R = Piv, R' = H (79%)
34 R = Piv, R' = Me (87%)
35 R = Me, R' = H (91%)
2 R = H, R' = H, R" = Ac
3 R = H, R' = H, R" = H
4 R = Me, R' = H, R" = Ac
5 R = H, R' = Me, R" = Ac
6 R = Me, R' = H, R" = H
7 R = Me, R' = Me, R" = Ac
i
i
j,k
Scheme 3. Synthesis of the disaccharides 2–7. Reaction conditions: (a) NIS, TfOH, MS 4 Å; (b) NaOMe, Guanidine; (c) DMSO, Ac2O; (d) L-selectride; (e) NaH, MeI; (f) NBS, CCl4;
(g) Bu3SnH, AIBN, Toluene; (h) NaOMe, MeOH; (i) KOH, EtOH; (j) Bu2SnO, MeOH; (k) MeI, CsF, DMF.
30, 31, and 32,22–24 which were then subjected to tributyltin hy-
dride/AIBN mediated radical reaction to afford 60-deoxy disaccha-
rides 33, 34, and 35.25 Transesterification gave the final
disaccharides 2, 5, and 4, respectively. The amino disaccharide
derivatives 3 and 6 were produced in quantitative yields after stir-
ring compounds 2 and 4 in ethanolic KOH solution at reflux. Meth-
ylation of compound 5 via stannyl acetal formation with dibutyltin
oxide in dry methanol, followed by addition of MeI, gave the di-O-
methylated disaccharide 7.26
g
¼ ISTD=I0 ꢁ ligand excess
The amplification factor
g is normalized by using the proton
with the highest saturation transfer set to 100% (Tyv H-6 protons
in this case), and all the amplification factors of other signals are
scaled to its percentage values. Greater
STD effect and closer contact with the protein. The STD epitope
mapping of all six compounds 2 to 7 in normalized
sented in Fig. 3 and Table 1,
g value indicates stronger
g values is pre-
2.2. Binding of disaccharides 2–7 to antibody IgG 9D4
2.3. STD effects observed for the tyvelose residue
Competitive enzyme linked immunosorbent assay (ELISA) was
used to determine IC50 values of disaccharides 2–7 for binding by
mAb 9D4.5 Briefly, the ELISA plate was coated with a synthetic
The tyvelose residue is mostly buried in the binding site. While
the whole sugar experiences moderate to high STD effects at all
positions, H-60 and H-50 protons appear to be the closest to the pro-
tein binding pocket. The weak saturation transfer to the methoxyl
group at C-20 is consistent with the positioning of this group at the
rim of the binding site. Tyvelose H-40 of compound 2 displays
strong saturation transfer, indicating its close proximity to the
antibody. Chemical mapping studies showed the 4-hydroxyl group
was an essential polar contact. Thus the hydroxyl group at C-40
forms a hydrogen bond with the binding site, and on the other
hand, the H-40 is in close contact with a hydrophobic pocket in
the antibody. The H-50 and H-60 of tyvelose ring also display strong
STD effects in all six compounds, suggesting both H-50 and H-60 are
involved in hydrophobic interactions with the binding site. To-
gether with H-40 and 40-hydroxyl group, H-50 and H-60 are envis-
aged to be buried in the binding site. H-10, H-20, and H-30 atoms
of the tyvelose epitope show moderate to strong STD effects indi-
cating close proximity to the surface of the site even though this
part of the tyvelose ring is not buried as deeply as the other half.
The antibody displayed ninefold higher affinity for the 2-O-meth-
ylated derivative 5 as compared to the affinity for the native
ligand 2, thus clearly suggesting the peripheral position of its 2-hy-
droxyl group. From this evidence it is concluded that the H-10, H-20,
and H-30 of tyvelose lie along a hydrophobic surface in the binding
[b-D-Tyvp-(1?3)-b-D-GalpNAc]8-BSA glycoconjugate, and antibody
was added to antigen coated plates in the presence of increasing
concentrations of synthetic disaccharide ligands. Bound antibody
was revealed by incubation with a horseradish peroxidase (HRP)-
labeled goat anti-rat IgG. Percent inhibition was calculated by
comparison with control wells without synthetic inhibitor. Com-
pared with the previously identified tighter binding ligands 3, 4,
and 5 (IC50 values in Fig. 3), containing single site modifications,
compounds 6 and 7 with IC50 values of 8 lM and 10 lM (Fig. 2)
did not result in increased binding affinity, despite pairing of the
favorable functional group changes.
In the STD NMR experiments, we wish to compare the binding
results of disaccharides 2–7, to see whether the differences in
binding activities measured by ELISA could be reflected by STD ef-
fects. The STD NMR spectrum and its reference spectrum for each
of the disaccharides 2 to 7 were obtained using similar parameters.
Only the resonances that could be unambiguously assigned were
used for calculating amplification factors g, the ratio of the inten-
sity of the signal in the difference spectrum (ISTD) and the corre-
sponding signal in the reference spectrum (I0), multiplied by
ligand excess.

4
L. Cui et al. / Carbohydrate Research 383 (2014) 1–13
experiences the strongest STD effect. All H-4 protons of the GalNAc
moiety of the six disaccharides demonstrate strong STD effects,
suggesting its close contact with the binding site. This is consistent
with the result from chemical mapping that steric clash at C-4
occurred when it was epimerized or O-methylated. The majority
of the binding energy at C-4 is therefore inferred to derive from
van der Waals’ interaction between H-4 and the binding site. This
is also supported by the chemical mapping, where the 4-deoxy
GalNAc derivative did not show a significant reduction in binding.
All six compounds experience moderate saturation transfer at
the H-6 of GalNAc, and the methyl groups at O-6 of compounds
4, 6, and 7 experience moderate to strong STD effects. Chemical
mapping inferred that C-6 is located at the periphery of the binding
site and STD effects support this conclusion. The enhancement in
binding energy by methylation at O-6 is realized by the introduc-
tion of hydrophobic interaction at the edge of the binding pocket.
The 6-O-methyl derivative 4 consistently shows stronger STD ef-
fects at most hydrogen atoms of the GalNAc ring and also C-30 of
tyvelose. This suggests that either disaccharide 4 binds more dee-
ply or in an altered orientation. Since nearly all protons experience
moderate to strong STD effects, disaccharide 4 must be bound in an
orientation that results in greater contact with binding site.
In the GalNAc epitope, the 2-acetamido group shows weak to
moderate STD effects in most of the compounds 2 to 7, which is
indicative that this group is not in close contact with the antibody.
Removal of the N-acetyl group resulted in a better ligand 3, as re-
vealed by chemical mapping. This leads to the conclusion that the
2-acetamido group of the native disaccharide ligand 2 is exposed to
bulk solvent, and its free amino sugar congener 3 achieves better
binding with the antibody possibly via water-mediated hydrogen
Figure 2. Inhibition curve of disaccharides 6 and 7 with rat monoclonal antibody
IgG 9D4. Each point was the average of triplicate measurements and the standard
deviation on each point was 5% or better.
pocket, while the 2-hydroxyl group stretches out of the binding
site, the edge of which is lipophilic, as inferred from the increased
binding affinity via the introduction of a 2-methoxy group.
2.4. STD effects observed for the GalNAc residue
The GalNAc moiety exhibits weak to moderate saturation trans-
fer effects. Since H-4 is in close contact with the protein, this group
Figure 3. Relative STD effects for disaccharides 2–7 interacting with monoclonal antibody IgG 9D4.

L. Cui et al. / Carbohydrate Research 383 (2014) 1–13
5
Table 1
Relative STD effects for disaccharides 2 to 7 interacting with mAb IgG 9D4
Proton position
Tyvelose residue
GlcNAc residue
H-10
H-20
H-30
H-40
H-50
H-60
C-20-OMe
H-1
H-2
H-3
H-4
H-5
H-6
NAc
C-1-OMe
C-6-OMe
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
—
76
—
—
87
—
70
78
73
59
82
69
57
70
84
49
63
51
85
—
—
—
—
—
115
93
—
89
105
—
100
100
100
100
100
100
NA
NA
NA
28
NA
39
57
53
85
54
51
58
66
42
96
55
45
67
60
43
93
—
72
69
90
85
85
85
87
90
57
—
85
53
66
62
55
66
74
52
55
55
38
NA
43
38
NA
43
32
33
32
31
29
33
NA
NA
72
NA
74
74
Protons that are not available in the molecule are labeled as NA; protons whose STD effects cannot be calculated due to spectral overlapping are labeled as ‘—’.
bonding. Other hydrogens of the GalNAc ring exhibit moderate STD
effects in all six compounds.
here are labeled as IUPAC nomenclature for carbohydrate mole-
cules. For some signals in 1H NMR spectra, the coupling patterns
were reported as multiplets due to high order coupling or signal
overlap. Mass spectrometry was performed under positive/nega-
tive-mode electrospray ionization (ESI) on a Micromass ZabSpec
Hybrid Sector-TOF. Microanalyses were performed by the
analytical services of the Department of Chemistry in University
of Alberta. Optical rotations were measured in a 10 cm cell with
a Perkin-Elmer 241 polarimeter at 22 2 °C and specific rotation
Combination of functional group modifications in compounds
6 and 7 did not result in additive free energy gains. STD NMR
suggested that could be explained by small relative movement
of the tyvelose and GalNAc residues. This probable conforma-
tional alteration was also observed for compound 4, as all of
its hydrogens along the GalNAc ring experience distinct and
stronger STD effects as compared to the other five compounds.
However, without coordinates for the protein residues of the
binding site, it is impossible to develop a quantitative interpreta-
tion of the data.
[a
]
D
values are given in units of 10ꢂ1 deg cm2 gꢂ1
, and the
concentrations are recorded in units of g (100 mL)ꢂ1
.
3.2. Enzyme-linked immunosorbent assays (ELISA)
2.5. Conclusions
Enzyme immunoassays were performed in triplicate in a 96-
well microtiter plate with monoclonal antibody IgG 9D4.(1) The
microtiter plates were coated by incubation with the disaccharide
A concise synthetic strategy to obtain disaccharides 2–7 was
developed. The binding studies of compounds 2–7 revealed the
disaccharide epitope is bound quite deeply in the antibody site,
and nearly all protons experience moderate to strong STD effects.
Chemical mapping showed mAb 9D4 recognizes the disaccharide
via H-bond with tyvelose O-40 and the 60-deoxy group buried in
the binding site with the C-2 position at the periphery of the site.
For the GalNAc residue there are no crucial polar contacts, but
C-4 and C-5 are in close contact with the protein and C-6 is located
at the periphery of the site and the 4-OH group likely accepts a
hydrogen bond from a solvent exposed binding site amino acid.
glycoconjugate, [b-
g/mL) at 4 °C overnight (18 h), then washed five times with
tween-PBS solution. Plates were patted dry, and blocked with a
phosphate buffered saline (PBS) solution of 2% BSA (100 L/well)
D-Tyvp-(1?3)-b-D-GalNAcp]8-BSA (100 lL/well,
1
l
l
for 1 h at rt. After washing the plate, a fixed volume of antibody
IgG 9D4 solution (100
l
L/well, at 1.0 ꢁ 10ꢂ4 dilution of ascites
fluid) containing increasing concentrations of disaccharide ligands
2 to 7 were added, and control wells containing no inhibitor were
used to establish the reference for IgG inhibition. After 24-hour
incubation at rt, the plate was washed, and a solution of goat
anti-rat IgG antibody conjugated with horse radish peroxidase
3. Experimental
(100 lL, 1:5000 dilution/well) was added and incubated for 1 h
at rt. The labeled secondary antibody was detected according to
the manufacturer’s suggestions (Kirkegaard and Perry Lab, Mandel,
Guelph, ON). The plate was washed, and 3,30,5,50-tetramethylbenz-
3.1. General methods
All chemical reagents were of analytical grade, and used as sup-
plied. Organic solvents in reactions were dried according to com-
mon literature protocols. All non-hydrolytic reactions were
conducted under a positive pressure of argon. Analytical thin layer
chromatography (TLC) was performed on silica gel 60-F254 (Merck).
Plates were visualized by ultraviolet (UV) light, or treatment with
ethanolic solution of sulfuric acid containing anisaldehyde, fol-
lowed by heating. Molecular sieves were stored in an oven
(>170 °C), and flame-dried in a flask under vacuum before use. Col-
idine solution (TMB, 100
was stopped after 2 min by addition of phosphoric acid (100
l
L/well) was added. The color reaction
L/
l
well, 1 M), and the blue solutions turned yellow. Absorbance was
detected at 450 nm, and percentage inhibition was calculated,
using wells without ligand as the reference for zero inhibition.
Inhibition curves and reported IC50 values were obtained with
the Microsoft Origin software, applying a four-parameter logistic
function fitting.
umn chromatography used silica gel (SiliCycle, F60, 40–63 lm,
3.3. Saturation transfer difference (STD) NMR
60 Å) and solvents were of reagent grade, and used as supplied.
High performance liquid chromatography (HPLC) was performed
using a Waters 2487 Dual k UV absorbance detector and separa-
tions were done on a Beckman C18 silica column. 1H NMR spectra
were recorded at 400, 500, or 600 MHz and 13C NMR spectra were
recorded at 100 or 125 MHz. First order chemical shifts are re-
ported in d (ppm) using residual solvent signals from deuterated
solvents as references. For samples in D2O, the chemical shifts
are referenced to external acetone (0.1% v/v) at d 2.225. Signals
in 1H and 13C NMR spectra were assigned with the aid of two
dimensional COSY, HMQC. and HMBC spectra. NMR data reported
An aqueous buffer solution of mAb IgG 9D4 was subjected to re-
peated ultrafiltration against PBS buffer made in D2O (pH 7.2). The
mAb in deuterated buffer was concentrated to 2.98 mg/mL as
determined by UV absorption (OD280). The protein solution
(700 lL, 2.98 mg/mL) and ligand solution (50 lL, 100-fold excess)
in PBS–D2O buffer were used throughout the NMR studies. The
STD NMR 1D experiments were done with a Varian Inova three-
channel 600 MHz spectrometer at 27 °C. The STD NMR epitope
mapping for the disaccharide ligands was achieved by applying a
series of equally spaced 50 ms Gaussian pulse for saturation, with

6
L. Cui et al. / Carbohydrate Research 383 (2014) 1–13
1 ms delay between pulses. The total saturation time was 1.53 s.
The frequency for protein irradiation was set to 7.8 ppm (absolute
frequency = 1650 Hz). The off-resonance irradiation was set at
33.0 ppm. Free induction decay (FID) values with on and off reso-
nance protein saturation were recorded alternatively, and their
subtraction was performed after each scan by phase cycling.
26.1((CH3)2C), 25.2((CH3)2C). Anal. Calcd for C12H20O5 (244.30): C,
59.00; H, 8.25. Found: C, 58.73; H, 8.41.
3.6. Phenyl 2,4,6-tri-O-acetyl-3-deoxy-1-thio-a,b-D-
glucopyranoside (11)
Protein resonances were suppressed by applying a protein T1
A solution of deoxy sugar 9 (16.50 g, 0.068 mol) in aqueous ace-
tic acid (60%, 200 mL) was warmed at 80 °C for 2 h and evaporated
to dryness. The residue was then dissolved in pyridine (80 mL) and
acetic anhydride (80 mL). The solution was stirred at rt. for 3 h and
concentrated to a syrup under reduced pressure. The syrup was
dissolved in ethyl acetate and extracted with water. The ethyl ace-
tate layer was dried over sodium sulfate, filtered, and evaporated
to give a clear syrup. The syrup in dichloromethane (200 mL) solu-
tion was cooled to 0 °C, followed by addition of thiophenol
(11.2 mL, 0.11 mol) and BF3ꢀOEt2 (12.5 mL, 0.10 mmol). The reac-
tion reached completion after 3 h and was neutralized by addition
of triethylamine. Column chromatography afforded thioglycoside
q
filter, a 0.03 s low power spin-lock pulse after the saturation pulse.
The resonance of residual HOD was reduced by a WATERGATE W5
pulse.27 The frequency of this irradiation was selected by an array
experiment. The saturation difference spectra were recorded over
2–4 k scans, while the reference spectra were recorded by 1–2 k
scans. The relative STD effect values were calculated by dividing
the intensities of signals in the difference spectrum by those in
the reference spectrum.
3.4. 1,2:5,6-di-O-Isopropylidene-3-O-
[(methylthio)thiocarbonyl]-
a
-D
-glucofuranose (8a)
11 (a:b = 1:1.3) as clear syrup (18.98 g, 73%, 3 steps). Data for the
a
isomer: ½a 2D2
ꢃ
+254.3 (c 2.6, CHCl3). 1H NMR (600 MHz, CDCl3): d
1,2:5,6-di-O-Isopropylidene-
a-D-glucofuranose (40.00 g, 0.15
7.50–7.46 (m, 2H, PhH), 7.33–7.25 (m, 3H, PhH), 5.80 (d, 1H,
J1,2 = 5.2 Hz, H-1), 5.10 (ddd, 1H, J2,3a = 12.9 Hz, J2,3e = 4.9 Hz, H-2),
4.86 (ddd, 1H, J4,3a = 11.0 Hz, J4,5 = 11.0 Hz, J4,3e = 4.9 Hz, H-4),
4.46 (ddd, 1H, J5,6a = 5.7 Hz, J5,6b = 2.3 Hz, H-5), 4.25 (dd, 1H,
J6a,6b = 12.1 Hz, H-6a), 4.12 (dd, 1H, H-6b), 2.39 (ddd, 1H,
J3e,3a = 11.9 Hz, H-3e), 2.11(s, 3H, OCOCH3), 2.08 (s, 3H, OCOCH3),
2.02 (s, 3H, OCOCH3), 1.93 (ddd, 1H, H-3a). 13C NMR (125 MHz,
CDCl3): d 170.7 (OCOCH3), 169.8 (OCOCH3), 169.6 (OCOCH3),
133.0 (Ph), 131.9 (Ph), 129.0 (Ph), 127.5 (Ph), 85.9 (C-1), 68.6 (C-
5), 67.9 (C-2), 66.0 (C-4), 62.5 (C-6), 30.9 (C-3), 20.9 (OCOCH3),
20.8 (OCOCH3), 20.7 (OCOCH3). Anal. Calcd for C18H22O7S
(382.43): C, 56.53; H, 5.80; S, 8.38. Found: C, 56.38; H, 5.98; S,
mol) was dissolved in dry THF (400 mL), followed by addition of
sodium hydride (15.00 g, 60% dispersion in mineral oil, 0.37 mol)
and imidazole (5.20 g, 0.075 mol). Vigorous bubbling occurred
when imidazole was added. The reaction was stirred for 2 h at rt,
and the colorless solution turned yellow. Carbon disulfide
(18.00 g, 0.30 mol) was then added and the reaction mixture was
kept at rt for 4 h, during which time it turned brownish yellow.
Iodomethane (19.2 mL, 0.30 mol) was added and the reaction
was completed in 1.5 h with formation of a white precipitate.
THF was then removed by evaporation. The residue dissolved in
ethyl acetate was extracted with water, dried over anhydrous so-
dium sulfate, and evaporated to dryness to afford a brownish yel-
low syrup. Column chromatography (1:30 ethyl acetate:hexanes)
8.27. Data for the b isomer: ½a D22
ꢃ
ꢂ9.2 (c 4.0, CHCl3). 1H NMR
gave clear syrup (53.80 g, 100%): ½a D22
ꢃ
ꢂ23.6 (c 8.1, CHCl3). 1H
(600 MHz, CDCl3): d 7.54–7.49 (m, 2H, PhH), 7.33–7.29 (m, 3H,
PhH), 4.84–4.78 (m, 2H, H-2, H-4), 4.67 (d, 1H, J1,2 = 9.8 Hz, H-1),
4.25 (dd, 1H, J6a,6b = 12.1 Hz, J6a,5 = 2.5 Hz, H-6a), 4.12 (dd, 1H,
J6b,5 = 6.0 Hz, H-6b), 4.46 (ddd, 1H, J5,4 = 9.8 Hz, H-5), 2.39 (ddd,
1H, J3e,3a = 12.0 Hz, J3e,2 = 5.0 Hz, J3e,4 = 5.0 Hz, H-3e), 2.10(s, 3H,
OCOCH3), 2.08 (s, 3H, OCOCH3), 2.04 (s, 3H, OCOCH3), 1.93 (ddd,
1H, J3a,2 = 11.6 Hz, J3a,4 = 11.6 Hz, H-3a). 13C NMR (125 MHz,
CDCl3): d 170.7 (OCOCH3), 169.4 (OCOCH3), 169.2 (OCOCH3),
132.6 (Ph), 132.5 (Ph), 128.8 (Ph), 128.0 (Ph), 87.3 (C-1), 78.0 (C-
5), 67.2 (C-2), 65.9 (C-4), 62.8 (C-6), 35.2 (C-3), 20.9 (OCOCH3),
20.8 (OCOCH3), 20.7 (OCOCH3). Anal. Calcd for C18H22O7S
(382.43): C, 56.53; H, 5.80; S, 8.38. Found: C, 56.69; H, 5.92; S, 8.49.
NMR (500 MHz, CDCl3): d 5.92 (app. d, 2H, J = 3.4 Hz, H-1, H-3),
4.68 (d, 1H, J = 3.9 Hz, H-2), 4.34 (dd, 1H, J4,5 = 7.5 Hz, J4,3 = 2.8 Hz,
H-4), 4.31 (ddd, 1H, J5,6a = 5.5 Hz, J5,6b = 5.5 Hz, H-5), 4.11 (dd, 1H,
J6a,6b = 8.8 Hz, H-6a), 4.06 (dd, 1H, H-6b), 2.60 (s, 3H, SCH3), 1.54
(s, 3H, (CH3)2C), 1.42 (s, 3H, (CH3)2C), 1.33 (s, 3H, (CH3)2C), 1.32
(s, 3H, (CH3)2C). 13C NMR (125 MHz, CDCl3): d 214.8 (CH3SCS–),
112.4 ((CH3)2C), 109.3 ((CH3)2C), 105.0 (C-1), 84.2 (C-2), 82.8 (C-
4), 79.8 (C-5), 72.4 (C-3), 67.0 (C-6), 26.8 ((CH3)2C), 26.7((CH3)2C),
26.3((CH3)2C), 25.3((CH3)2C), 19.3(SCH3). Anal. Calcd for
C14H22O6S2(350.50): C, 47.98; H, 6.33; S, 18.30. Found: C, 47.67;
H, 6.11; S, 18.08.
3.7. Phenyl 4,6-O-benzylidene-3-deoxy-1-thio-a,b-D-
glucopyranoside (13)
3.5. 3-Deoxy-1,2:5,6-di-O-isopropylidene-a-D-ribo-
hexofuranose (9)
The
a/b mixture of compound 11 (a:b = 1:1.3, 10.00 g,
To a solution of compound 8a (50.52 g, 0.14 mol) in toluene
(500 mL) was added a catalytic amount of azobisisobutyronitrile
(AIBN) and tributyltin hydride (45.8 mL, 0.17 mol). The reaction
was heated at reflux for 5 h. The solution was concentrated by dis-
tillation, and the residue was loaded on a silica gel column, which
was eluted first with hexanes to remove the tin derivatives. Col-
umn chromatography (1:10 acetone:hexanes) afforded the deoxy
0.026 mol) was suspended in dry methanol (100 mL). Sodium
methoxide in methanol solution (1.7 M, 3.1 mL, 5.2 mmol) was
added, and the reaction mixture was stirred at rt. for 1 h. Dowex
50WX4-50 H+ resin was added to neutralize the solution, and the
resin was removed by filtration. The filtrate was evaporated to dry-
ness and the residue was dissolved in dry distilled acetonitrile
(60 mL), followed by addition of benzaldehyde dimethylacetal
(7.8 mL, 0.052 mol) and toluenesulfonic acid (0.90 g, 5.2 mmol).
After stirring at rt for 6 h, the reaction was neutralized by addition
of triethylamine. The residue after evaporation was dissolved in
ethyl acetate and extracted with water. Column chromatography
sugar as a clear syrup (26.41 g, 75%): ½a D22
ꢃ
ꢂ7.6 (c 1.8, CHCl3). 1H
NMR (500 MHz, CDCl3): d 5.78 (d, 1H, J1,2 = 3.7 Hz, H-1), 4.72 (dd,
1H, J2,3a = 4.3 Hz, H-2), 4.14–4.05 (m, 3H, H-4, H-5, H-6a), 3.79
(dd, 1H, J6b,6a = 7.5, J6b,5 = 4.8 Hz, H-6b), 2.15 (dd, 1H, J3e,3a = 13.4 -
Hz, J3e,4 = 4.4 Hz, H-3e), 1.73 (ddd, 1H, J3a,4 = 10.3 Hz, H-3a), 1.48
(s, 3H, (CH3)2C), 1.39 (s, 3H, (CH3)2C), 1.32 (s, 3H, (CH3)2C), 1.28
(s, 3H, (CH3)2C). 13C NMR (125 MHz, CDCl3): d 111.3 ((CH3)2C),
109.6 ((CH3)2C), 105.6 (C-1), 80.4 (C-2), 78.6 (C-4), 76.8 (C-5),
67.2 (C-6), 35.3 (C-3), 26.8 ((CH3)2C), 26.5((CH3)2C),
(1:50 acetone/toluene) gave 13 (a:b = 1:1.3) as a white solid
(8.60 g, 96%, 2 steps). Data for the
a
isomer: ½a 2D2
ꢃ
+322.0 (c 2.4,
CHCl3). 1H NMR (500 MHz, CDCl3): d 7.56–7.50 (m, 4H, PhH),
7.41–7.29 (m, 6H, PhH), 5.56 (s, 1H, PhCH), 5.52 (d, 1H,
J1,2 = 5.0 Hz, H-1), 4.32 (dd, 1H, J6e,6a = 10.3 Hz, J6e,5 = 4.9 Hz,

L. Cui et al. / Carbohydrate Research 383 (2014) 1–13
7
H-6e), 4.19 (ddd, 1H, J5,4 = 9.7 Hz, J5,6a = 9.7 Hz, H-5), 4.11 (m, 1H,
H-2), 3.77 (dd, 1H, H-6a), 3.62 (ddd, 1H, J4,3a = 13.4 Hz,
J4,3e = 4.1 Hz, H-4), 2.42 (ddd, 1H, J3e,2 = 4.2 Hz, J3e,3a = 11.7 Hz, H-
3e), 2.26 (d, 1H, JOH,2 = 10.1 Hz, OH), 1.78 (ddd, 1H, J3a,2 = 11.8 Hz,
H-3a). 13C NMR (125 MHz, CDCl3): d 137.2 (Ph), 133.8 (Ph), 132.2
(Ph), 129.2 (Ph), 128.4 (Ph), 127.8 (Ph), 126.2 (Ph), 101.9 (PhCH),
93.5 (C-1), 76.1 (C-4), 69.1 (C-6), 67.8 (C-2), 65.9 (C-5), 35.3 (C-
3). Anal. Calcd for C19H20O4S (344.43): C, 66.26; H, 5.85; S, 9.31.
layer was dried over Na2SO4, filtered, and the ethyl ether was evap-
orated to give oxazoline as a brown syrup. The resulting interme-
diate (66 g, 0.26 mol) was redissolved in dry methanol (400 mL),
and the pH of the solution was adjusted to 2 with p-toluenesul-
fonic acid. The reaction mixture was stirred for 4 h, and neutralized
with Et3N. Methanol was evaporated, and column chromatography
(1:10 methanol/dichloromethane) gave a white solid (51.44 g,
81%): ½a 2D2
ꢃ
ꢂ30.8 (c 5.6, CH3OH). 1H NMR (500 MHz, D2O): d 4.45
Found: C, 66.01; H, 5.92; S, 9.42. Data for the b isomer: ½a D22
ꢃ
(d, 1H, J1,2 = 8.5 Hz, H-1), 3.94 (dd, 1H, J6a,6b = 12.3 Hz,
J6a,5 = 1.9 Hz, H-6a), 3.75 (dd, 1H, J6b,5 = 5.6 Hz, H-6b), 3.69 (dd,
1H, J2,3 = 10.3 Hz, H-2), 3.54 (dd, 1H, J3,4 = 8.6 Hz, H-3), 3.51 (s,
3H, OCH3), 3.42–3.48 (m, 2H, H-4, H-5), 2.04 (s, 3H, NHCOCH3).
13C NMR (125 MHz, CDCl3): d 175.6 (NHCOCH3), 102.8 (C-1), 76.8
(C-3), 74.8 (C-5), 70.8 (C-4), 61.6 (C-6), 57.9 (OCH3), 56.3 (C-2),
23.0 (NHCOCH3). ES HRMS calcd for C9H17NO6Na (M+Na):
258.0948, found: 258.0949.
ꢂ43.0 (c 1.6, CHCl3). 1H NMR (500 MHz, CDCl3): d 7.56–7.53
(m, 2H, PhH), 7.50–7.47 (m, 2H, PhH), 7.39–7.33 (m, 6H, PhH),
5.54 (s, 1H, PhCH), 4.57 (d, 1H, J1,2 = 9.5 Hz, H-1), 4.75 (dd, 1H,
J6e,6a = 10.6 Hz, J6e,5 = 4.9 Hz, H-6e), 3.78 (dd, 1H, J5,6a = 10.3 Hz, H-
6a), 3.62–3.58 (m, 2H, H-2, H-4), 3.49 (ddd, 1H, J5,4 = 9.8 Hz, H-5),
2.59 (ddd, 1H, J3e,3a = 11.9 Hz, J3e,2 = 4.6 Hz, J3e,4 = 4.6 Hz, H-3e),
2.45 (d, 1H, JOH,2 = 2.1 Hz, OH), 1.78 (ddd, 1H, J3a,2 = 11.6 Hz,
J3a,4 = 11.6, H-3a). 13C NMR (125 MHz, CDCl3): d 137.2 (Ph), 132.7
(Ph), 131.7 (Ph), 129.2 (Ph), 129.1 (Ph), 128.3 (Ph), 126.2 (Ph),
101.8 (PhCH), 91.6 (C-1), 75.9 (C-4), 74.0 (C-5), 69.0 (C-6), 67.5
(C-2), 36.7 (C-3). Anal. Calcd for C19H20O4S (344.43): C, 66.26; H,
5.85; S, 9.31. Found: C, 66.19; H, 6.03; S, 9.40.
3.10. Methyl 2-acetamido-2-deoxy-3,6-di-O-pivaloyl-b-D-
glucopyranoside (18)
To a solution of compound 17 (4.30 g, 18 mmol) in dry dichloro-
methane (20 mL) and pyridine (20 mL) at 0 °C was added pivaloyl
chloride (3 mL, 54 mmol) dropwise over 10 min. The reaction mix-
ture was stirred at 0 °C for 2 h, and evaporated to dryness. Column
chromatography (1:8 acetone/toluene) gave 18 as a white solid
3.8. Phenyl 2-O-acetyl-4,6-O-benzylidene-3-deoxy-1-thio-a,b-D-
glucopyranoside (14)
The
a/b mixture of compound 13 (a:b = 1:1.3, 2.00 g, 5.8 mmol)
(6.66 g, 90%): ½a D22
ꢃ
ꢂ43.07 (c 0.95, CHCl3). 1H NMR (500 MHz,
was dissolved in pyridine: acetic anhydride solution (20 mL, 1:1).
After 1 h, the pyridine and acetic anhydride were co-evaporated
with toluene, and the residue was loaded on a silica gel column
and eluted with acetone/hexanes (1:10) to afford a white solid
CDCl3): d 5.68 (d, 1H, JNH,2 = 7.6 Hz, NHCOCH3), 5.04 (dd, 1H,
J3,4 = 10.6 Hz, J3,2 = 8.7 Hz, H-3), 4.44–4.36 (m, 3H, H-1, H-6a, H-
6b), 3.97 (ddd, 1H, J2,1 = 8.8 Hz, H-2), 3.57–3.49 (m, 2H, H-4, H-5),
3.47 (s, 3H, OCH3), 1.94 (s, 3H, NHCOCH3), 1.24 ((CH3)3CCO–), 1.20
((CH3)3CCO–). 13C NMR (125 MHz, CDCl3): d 179.9 ((CH3)3CCO–),
179.2 ((CH3)3CCO–), 170.0 (NHCOCH3), 102.0 (C-1), 75.0 (C-3),
74.3 (C-5), 69.4 (C-4), 63.2 (C-6), 56.4 (OCH3), 53.7 (C-2), 39.0
((CH3)3CCO–), 38.9 ((CH3)3CCO–), 27.2 ((CH3)3CCO–), 27.0
((CH3)3CCO–), 23.3 (NHCOCH3). Anal. Calcd for C19H33NO8
(403.47): C, 56.56; H, 8.24; N, 3.47. Found: C, 56.17; H, 8.18; N, 3.85.
14 (
a
:b = 1:1.3, 2.20 g, 98%). Data for the
a
isomer: ½a 2D2
ꢃ
+264.5
(c 4.6, CHCl3). 1H NMR (600 MHz, CDCl3): d 7.55–7.45 (m, 4H,
PhH), 7.40–7.28 (m, 6H, PhH), 5.81 (d, 1H, J1,2 = 5.3 Hz, H-1), 5.57
(s, 1H, PhCH), 5.16 (ddd, 1H, J2,3a = 12.3 Hz, J2,3e = 4.9 Hz, H-2),
4.32 (ddd, 1H, J5,6a = 9.9 Hz, J5,4 = 9.9 Hz, J5,6e = 4.8 Hz, H-5), 4.22
(dd, 1H, J6e,6a = 10.3 Hz, H-6e), 3.75 (dd, 1H, H-6a), 3.70 (ddd, 1H,
J4,3a = 11.8 Hz, J4,3e = 4.3 Hz, H-4), 2.33 (ddd, 1H, J3e,3a = 11.5 Hz, H-
3e), 2.13 (s, 3H, OCOCH3), 2.13 (ddd, 1H, H-3a). 13C NMR
(100 MHz, CDCl3): d 170.0 (OCOCH3), 137.2 (Ph), 133.2 (Ph),
132.2 (Ph), 129.2 (Ph), 129.1 (Ph), 128.4 (Ph), 127.5 (Ph), 126.2
(Ph), 101.9 (PhCH), 86.6 (C-1), 76.1 (C-4), 69.0 (C-6), 68.8 (C-2),
65.0 (C-5), 31.2 (C-3), 21.0 (OCOCH3). Anal. Calcd for C21H22O5S
(386.46): C, 65.27; H, 5.74; S, 8.30. Found: C, 65.29; H, 5.56; S,
3.11. Methyl 2-acetamido-2-deoxy-4,6-dipivaloyl-O-b-D-
galactopyranoside (19)
Compound 18 (3.70 g, 9.2 mmol) was dissolved in dry distilled
dichloromethane (37 mL), and dry pyridine (3.7 mL) was added.
The solution was then cooled to ꢂ35 °C, and trifluoromethanesul-
fonic anhydride (1.7 mL, 10 mmol) was added dropwise. The mix-
ture was then allowed to warm to rt. over 1 h, and water (6.3 mL)
was added. The reaction was refluxed for 4 h, and stirred at rt over-
night. After concentration, column chromatography (1:8 acetone/
8.77. Data for the b isomer: ½a D22
ꢃ
ꢂ25.3 (c 14.8, CHCl3). 1H NMR
(600 MHz, CDCl3): d 7.55–7.42 (m, 4H, PhH), 7.40–7.28 (m, 6H,
PhH), 5.54 (s, 1H, PhCH), 4.88 (ddd, 1H, J2,3a = 10.9 Hz,
J2,1 = 9.9 Hz, J2,3e = 5.0 Hz, H-2), 4.75 (d, 1H, H-1), 4.37 (dd, 1H,
J6e,6a = 10.6 Hz, J6e,5 = 4.9 Hz, H-6e), 3.78 (dd, 1H, J6a,5 = 10.4 Hz, H-
6a), 3.63 (ddd, 1H, J4,3a = 11.8 Hz, J4,5 = 9.1 Hz, J4,3e = 4.2 Hz, H-4),
3.50 (ddd, 1H, H-5), 2.59 (ddd, 1H, J3e,3a = 11.6 Hz, H-3e), 2.12 (s,
3H, OCOCH3), 1.81 (ddd, 1H, H-3a). 13C NMR (100 MHz, CDCl3): d
170.0 (OCOCH3), 137.2 (Ph), 133.2 (Ph), 132.2 (Ph), 129.2 (Ph),
129.1 (Ph), 128.4 (Ph), 127.5 (Ph), 126.2 (Ph), 101.9 (PhCH), 86.6
(C-1), 76.1 (C-4), 69.0 (C-6), 68.8 (C-2), 65.0 (C-5), 31.2 (C-3),
21.0 (OCOCH3). Anal. Calcd for C21H22O5S (386.46): C, 65.27; H,
5.74; S, 8.30. Found: C, 65.27; H, 5.75; S, 8.45.
toluene) afforded compound 19 as a white solid (3.3 g, 89%): ½a D22
ꢃ
ꢂ28.0 (c 2.10, CHCl3). 1H NMR (600 MHz, CDCl3): d 5.69 (d, 1H,
JNH,2 = 4.2 Hz, NHCOCH3), 5.31 (d, 1H, J4,3 = 3.3 Hz, H-4), 4.45 (d,
1H, J1,2 = 8.2 Hz, H-1), 4.18 (dd, 1H, J6a,6b = 11.2 Hz, J6a,5 = 7.4 Hz,
H-6a), 4.11 (dd, 1H, J6b,5 = 6.1 Hz, H-6b), 4.00 (dd, 1H,
J3,2 = 10.4 Hz, H-3), 3.90 (dd, 1H, H-5), 3.67 (ddd, 1H, H-2), 3.53
(s, 3H, OCH3), 2.07 (s, 3H, NHCOCH3), 1.27 ((CH3)3CCO–), 1.20
((CH3)3CCO–). 13C NMR (125 MHz, CDCl3): d 178.0 ((CH3)3CCO–),
177.8 ((CH3)3CCO–), 173.1 (NHCOCH3), 101.2 (C-1), 72.1 (C-3),
71.5 (C-5), 68.3 (C-4), 61.9 (C-6), 56.7 (OCH3), 55.8 (C-2), 39.2
((CH3)3CCO–), 38.7 ((CH3)3CCO–), 27.2 ((CH3)3CCO–), 27.1
((CH3)3CCO–), 23.5 (NHCOCH3). ES HRMS calcd for C19H34NO8Na
(M+Na): 404.2279, found: 404.2269.
3.9. Methyl 2-acetamido-2-deoxy-b-D-glucopyranoside (17)
Anhydrous iron(III) chloride (90.00 g, 0.56 mol) was added to a
suspension of 2-acetamido-2-deoxy- -glucose (60.00 g, 0.27 mol)
D
in dry distilled acetone (1.2 L). The mixture was stirred and heated
at reflux for 20 min under argon. The solution was cooled to 0 °C,
and Et3N (294 mL) and acetone (800 mL) were added with stirring.
The reaction was quenched with a solution of Na2CO3 (800 mL,
1.5 M), concentrated, and extracted with ethyl ether. The organic
3.12. Methyl 2-acetamido-2-deoxy-6-O-methyl-b-D-
glucopyranoside (20)
Compound 17 (5.00 g, 0.021 mol) was suspended in dry toluene
(200 mL), and bis(tributyltin) oxide (20 mL, 0.034 mol) was added

8
L. Cui et al. / Carbohydrate Research 383 (2014) 1–13
slowly. The reaction mixture was heated at reflux for 2 h, and the
solution turned clear, indicating complete consumption of starting
material. The solvent was then evaporated under reduced pressure
to afford a brownish syrup, to which methyl iodide (50 mL) was
added. The solution was stirred at reflux for 48 h and concentrated.
Column chromatography (2:1 acetone/toluene) produced com-
3.15. Methyl 2-acetamido-3-O-(2-O-acetyl-4,6-O-benzylidene-3-
deoxy-b- -ribo-hexopyranosyl)-2-deoxy-4,6-di-O-pivaloyl-b-
galactopyranoside (23)
D
D-
Monosaccharide acceptor 19 (2.0 g, 5.0 mmol) and thioglycoside
donor 14 ( :b = 1:1.3, 2.5 g, 6.4 mmol) were dissolved in dry dichlo-
a
pound 20 as a white solid (4.08 g, 78%): ½a D22
ꢃ
ꢂ24.7 (c 3.11, CH3OH).
romethane (100 mL). Molecular sieves (20.00 g, 4 Å) were added
and the mixture was stirred overnight at room temperature under
argon. To the stirred solution was added N-iodosuccinimide
(1.7 g, 7.5 mmol) and the reaction was cooled to 0 °C. After addition
of triflic acid (0.25 mL) over 5 min, the reaction mixture turned red,
and was then stirred for 2 h at 0 °C at which point the reaction
reached completion. Triethylamine (1 mL) was then added to neu-
tralize the solution. Column chromatography (1:4 acetone/toluene)
gave the title disaccharide 23 as a white solid (b only, 2.9 g, 85%):
1H NMR (600 MHz, CD3OD): d 4.29 (d, 1H, J1,2 = 8.4 Hz, H-1), 3.72
(dd, 1H, J6a,6b = 10.9 HzHHH, J6a,5 = 2.1 Hz, H-6a), 3.61 (dd, 1H,
J2,3 = 10.4 Hz, H-2), 3.58 (dd, 1H, J6b,5 = 5.8 Hz, H-6b), 3.43 (s, 3H,
C-1-OCH3), 3.41 (dd, 1H, J3,4 = 8.7 Hz, H-3), 3.39 (s, 3H, C-6-
OCH3), 3.36 (ddd, 1H, J5,4 = 9.9 Hz, H-5), 3.30 (m, 1H, H-4), 1.96
(s, 3H, NHCOCH3). 13C NMR (125 MHz, CD3OD):
d 170.4
(NHCOCH3), 100.2 (C-1), 73.5 (C-3), 72.8 (C-5), 69.8 (C-6), 68.8
(C-4), 56.3 (C-6-OCH3), 53.9 (C-2), 53.6 (C-1-OCH3), 20.0
(NHCOCH3). Anal. Calcd for C10H19NO6 (249.12): C, 48.19; H,
7.68; N, 5.62. Found: C, 47.76; H, 7.50; N, 5.62.
½
a 2D2 +1.1 (c 2.14, CHCl3). 1H NMR (500 MHz, CDCl3): d 7.44–7.43
(m, 2H, PhH), 7.39–7.32 (m, 3H, PhH), 5.59 (d, 1H, JNH,2 = 7.1 Hz,
ꢃ
NHCOCH3), 5.52 (s, 1H, PhCH), 5.40 (d, 1H, J4,3 = 3.2 Hz, H-4), 4.96
0
0
0
0
(d, 1H, J1,2 = 8.3 Hz, H-1), 4.72 (ddd, 1H, J2 ,3a = 10.0 Hz, J2 ,1 = 8.3 Hz,
3.13. Methyl 2-acetamido-2-deoxy-6-O-methyl-3-O-pivaloyl-b-
J2 ,3e = 5.5 Hz, H-20), 4.67 (dd, 1H, J3,2 = 10.8 Hz, H-3), 4.62 (d, 1H, H-
0
0
D-glucopyranoside (21)
10), 4.29 (dd, 1H, J6e ,6a = 10.6 Hz, J6e ,5 = 4.9 Hz, H-6e0), 4.12 (dd, 1H,
J6a,6b = 11.0 Hz, J6a,5 = 5.6 Hz, H-6a), 4.04 (dd, 1H, J6b,5 = 7.4 Hz, H-
0
0
0
0
Compound 20 (2.00 g, 8.0 mmol) was dissolved in dichloro-
6b), 3.92 (dd, 1H, H-5), 3.73 (dd, 1H, J6a ,5 = 10.4 Hz, H-6a0), 3.61
0
0
methane:pyridine (20 mL, 1:1), the solution was cooled to 0 °C,
and pivaloyl chloride (1.1 mL, 8.8 mmol) was added. The reaction
mixture was allowed to warm to ambient temperature over 2 h,
and then evaporated to dryness. Column chromatography (1:2 ace-
(ddd, 1H, J4 ,3a = 11.5 Hz, J4 ,5 = 9.1 Hz, J4 ,3e = 4.4 Hz, H-40), 3.51 (s,
0
0
0
0
0
0
3H, OCH3), 3.44 (ddd, 1H, H-50), 3.26 (ddd, 1H, H-2), 2.47 (ddd,
1H, J3e ,3a = 11.5 Hz, H-3e0), 2.07 (s, 3H, OCOCH3), 2.01 (s, 3H,
NHCOCH3), 1.68 (ddd, 1H, H-3a0), 1.23 (s, 9H, (CH3)3CCO–), 1.21 (s,
9H, (CH3)3CCO–). 13C NMR (125 MHz, CDCl3): d 177.9 ((CH3)3CCO–),
176.9 ((CH3)3CCO–), 170.8 (NHCOCH3), 169.3 (OCOCH3), 137.2
(Ph), 129.1 (Ph), 128.3 (Ph), 126.1 (Ph), 102.1 (C-10), 101.6 (PhCH),
100.2 (C-1), 75.0 (C-40), 74.4 (C-3), 71.4 (C-5), 70.1 (C-50), 70.0 (C-
20), 69.0 (C-60), 68.6 (C-4), 62.2 (C-6), 57.0 (OCH3), 55.1 (C-2), 39.1
((CH3)3CCO–), 38.7 ((CH3)3CCO–), 33.2 (C-30), 27.2 ((CH3)3CCO–),
27.1 ((CH3)3CCO–), 23.7 (NHCOCH3), 21.0 (OCOCH3). ES HRMS calcd
for C34H49NO13Na (M+Na): 702.30961, found: 702.30965. Anal.
Calcd for C34H49NO13(679.32): C, 60.08; H, 7.27; N, 2.06. Found: C,
59.97; H, 7.50; N, 2.25.
0
0
tone/toluene) afforded 21 as a white solid (2.29 g, 86%): ½a D22
ꢃ
ꢂ61.83 (c 2.10, CHCl3). 1H NMR (500 MHz, CDCl3): d 5.37 (d, 1H,
JNH,2 = 9.6 Hz, NHCOCH3), 4.98 (dd, 1H, J3,2 = 10.7 Hz, J3,4 = 9.1 Hz,
H-3), 4.39 (d, 1H, J2,1 = 8.3 Hz, H-1), 4.01 (ddd, 1H, H-2), 3.79–
3.72 (m, 2H, H-4, H-6a), 3.68 (dd, 1H, J6b,6a = 10.2 Hz,
J6b,5 = 4.8 Hz, H-6b), 3.50 (ddd, 1H, J5,4 = 10.4 Hz, J5,6a = 4.8 Hz, H-
5), 3.49 (s, 3H, C-1-OCH3), 3.43 (s, 3H, C-6-OCH3), 1.94 (s, 3H,
NHCOCH3), 1.21 ((CH3)3CCO–). 13C NMR (125 MHz, CDCl3): d
179.8 ((CH3)3CCO–), 170.2 (NHCOCH3), 102.0 (C-1), 75.4 (C-3),
74.3 (C-5), 72.2 (C-6), 70.7 (C-4), 59.6 (C-6-OCH3), 56.5 (C-1-
OCH3), 53.6 (C-2), 39.0 ((CH3)3CCO–), 27.0 ((CH3)3CCO–), 23.2
(NHCOCH3). Anal. Calcd for C15H27NO7 (333.38): C, 54.04; H,
8.16; N, 4.20. Found: C, 53.85; H, 8.12; N, 4.07.
3.16. Methyl 2-acetamido-3-O-(2-O-acetyl-4,6-O-benzylidene-3-
deoxy-b-
D
-ribo- hexopyranosyl)-2-deoxy-6-O-methyl-4-O-
pivaloyl-b-
D-galactopyranoside (24)
3.14. Methyl 2-acetamido-2-deoxy-6-O-methyl-4-O-pivaloyl-b-
Monosaccharide acceptor 22 (0.50 g, 1.5 mmol) and thioglyco-
side donor 14 ( :b = 1:1.3, 1.16 g, 3.0 mmol) were dissolved in dry
D-galactopyranoside (22)
a
dichloromethane (15 mL). Molecular sieves (3.00 g, 4 Å) were
added and the mixture was stirred overnight at room temperature
under argon. To the stirred solution was added N-iodosuccinimide
(0.71 g, 3.2 mmol) and the reaction was cooled to 0 °C. After addi-
Compound 21 (1.00 g, 3.0 mmol) was dissolved in dry distilled
dichloromethane (10 mL), and dry pyridine (1.0 mL) was added.
The solution was then cooled to ꢂ35 °C, and trifluoromethanesul-
fonic anhydride (0.56 mL, 3.3 mmol) was added dropwise. The
mixture was then allowed to warm to rt. over 1 h, and water
(1.0 mL) was added. The reaction was refluxed for 4 h, and stirred
at rt. for 18 h. After concentration, column chromatography (1:2
acetone/toluene) afforded compound 22 as a white solid (0.92 g,
tion of triflic acid (40 lL) over 3 min, the reaction mixture turned
red, and was stirred for 3 h at 0 °C. Triethylamine was added to neu-
tralize the solution, and the mixture was applied to column chro-
matography (1:4 acetone/toluene) to give the title disaccharide
24 as a white solid (b only, 0.69 g, 75%): ½a D22
ꢃ
+1.64 (c 2.14, CHCl3).
92%): ½a 2D2
ꢃ
ꢂ10.5 (c 0.76, CHCl3). 1H NMR (600 MHz, CDCl3): d
1H NMR (500 MHz, CDCl3): d 7.50–7.43 (m, 2H, PhH), 7.40–7.32 (m,
3H, PhH), 5.61 (d, 1H, JNH,2 = 7.2 Hz, NHCOCH3), 5.51 (s, 1H, PhCH),
5.38 (d, 1H, J4,3 = 3.3 Hz, H-4), 4.94 (d, 1H, J1,2 = 8.4 Hz, H-1), 4.73
5.73 (d, 1H, JNH,2 = 3.0 Hz, NHCOCH3), 5.30 (d, 1H, J4,3 = 3.2 Hz, H-
4), 4.44 (d, 1H, J1,2 = 8.4 Hz, H-1), 3.96 (dd, 1H, J3,2 = 10.4 Hz, H-3),
3.80 (dd, 1H, J5,6a = 5.8 Hz, J5,6b = 5.8 Hz, H-5), 3.71 (ddd, 1H, H-2),
3.55 (s, 3H, C-1-OCH3), 4.50 (dd, 1H, J6a,6b = 10.2 Hz, H-6a), 3.46
(dd, 1H, H-6b), 3.35 (s, 3H, C-1-OCH3), 2.07 (s, 3H, NHCOCH3),
(ddd, 1H, J2 ,3a = 10.8 Hz, J2 ,1 = 7.3 Hz, J2 ,3e = 5.6 Hz, H-20), 4.62 (d,
0
0
0
0
0
0
1H, H-10), 4.60 (dd, 1H, J3,2 = 10.8 Hz, H-3), 4.31 (dd, 1H, J6e ,6a
0
0
= 10.6 Hz, J6e ,5 = 5.0 Hz, H-6e0), 3.82 (dd, 1H, J5,6a = 5.8 Hz,
1.28 ((CH3)3CCO–). 13C NMR (125 MHz, CDCl3):
d
178.1
0
0
J5,6b = 5.8 Hz, H-5), 3.72 (dd, 1H, J6a ,5 = 10.3 Hz, H-6a0), 3.60 (ddd,
0
0
((CH3)3CCO–), 173.1 (NHCOCH3), 101.2 (C-1), 73.1 (C-5), 72.3
(C-3), 71.3 (C-6), 69.2 (C-4), 59.3 (C-6-OCH3), 56.6 (C-1-OCH3),
55.5 (C-2), 39.3 ((CH3)3CCO–), 27.3 ((CH3)3CCO–), 23.4 (NHCOCH3).
Anal. Calcd for C15H27NO7 (333.38): C, 54.04; H, 8.16; N, 4.20.
Found: C, 53.99; H, 8.18; N, 4.08.
1H, J4 ,3a = 11.5 Hz, J4 ,5 = 10.2 Hz, J4 ,3e = 4.6 Hz, H-40), 3.53 (s, 3H,
0
0
0
0
0
0
C-1-OCH3), 3.50–3.39 (m, 3H, H-50, H-6a, H-6b), 3.35 (s, 3H, C-6-
0
0
OCH3), 3.34–3.30 (m, 1H, H-2), 2.47 (ddd, 1H, J3e ,3a = 11.8 Hz, H-
3e0), 2.07 (s, 3H, OCOCH3), 2.00 (s, 3H, NHCOCH3), 1.68 (ddd, 1H,

L. Cui et al. / Carbohydrate Research 383 (2014) 1–13
9
H-3a0), 1.23 (s, 9H, (CH3)3CCO–). 13C NMR (125 MHz, CDCl3): d 177.1
3.19. Methyl 2-acetamido-3-O-(4,6-O-benzylidene-3-deoxy-b-D-
((CH3)3CCO–), 170.7 (NHCOCH3), 169.4 (OCOCH3), 137.2 (Ph), 129.1
(Ph), 128.3 (Ph), 126.1 (Ph), 101.9 (C-10), 101.6 (PhCH), 100.3 (C-1),
75.0 (C-3), 74.7 (C-40), 72.9 (C-5), 71.5 (C-6), 70.2 (C-50), 79.9 (C-20),
69.1 (C-4), 69.0 (C-60), 59.3 (C-6-OCH3), 57.0 (C-1-OCH3), 55.3 (C-2),
39.1 ((CH3)3CCO–), 33.3 (C-30), 27.3 ((CH3)3CCO–), 23.7 (NHCOCH3),
21.0 (OCOCH3). Anal. Calcd for C30H43NO12(609.66): C, 59.10; H,
7.11; N, 2.30. Found: C, 59.54; H, 7.49; N, 2.30.
ribo-hexopyranosyl)- 2-deoxy-6-O-methyl-4-O-pivaloyl-b-D-
galactopyranoside (26)
Compound 24 (0.30 g, 0.49 mmol) was dissolved in guanidine
solution (3.0 mL), and was stirred for 24 h under argon. The solu-
tion was evaporated to dryness, followed by column chromatogra-
phy (acetone/toluene, 1:3) to give a white solid (0.27 g, 0.47 mmol,
95%): ½a 2D2
ꢃ
+5.2 (c 1.80, CHCl3). 1H NMR (500 MHz, CDCl3): d 7.50–
7.45 (m, 2H, PhH), 7.40–7.32 (m, 3H, PhH), 5.74 (d, 1H, JNH,2 = 7.5 -
3.17. Methyl 2-acetamido-3-O-(4,6-O-benzylidene-3-deoxy-b-
D-
ribo-hexopyranosyl)-2- deoxy-4,6-di-O-pivaloyl-b-D-
galactopyranoside (25)
Hz, NHCOCH3), 5.50 (s, 1H, PhCH), 5.35 (d, 1H, J4,3 = 3.0 Hz, H-4),
4.65 (d, 1H, J1,2 = 8.3 Hz, H-1), 4.37 (d, 1H, J1 ,2 = 7.5 Hz, H-10),
0
0
4.28 (dd, 1H, J6e ,6a = 10.6 Hz, J6e ,5 = 5.0 Hz, H-6e0), 4.26 (dd, 1H,
J3,2 = 10.6 Hz, H-3), 3.84–3.70 (m, 3H, H-40, H-2, H5), 3.53 (s, 3H,
C-1-OCH3), 3.52–3.43 (m, 4H, H-6a0, H-20, H-6a, H-6b), 3.39 (ddd,
0
0
0
0
Compound 23 (2.1 g, 3.1 mmol) was dissolved in guanidine solu-
tion (20 mL), and was stirred for 48 h under argon. The reaction was
monitored by TLC, and the solution was evaporated to dryness. Col-
umn chromatography (1:4 acetone/toluene) gave a white solid 25
1H, J5 ,4 = 9.6 Hz, J5 ,6a = 9.6 Hz, H-50), 3.35 (s, 3H, C-6-OCH3), 2.41
0
0
0
0
(ddd, 1H, J3e ,3a = 11.8 Hz, J3e ,2 = 4.7 Hz, J3e ,4 = 4.7 Hz, H-3e0), 2.01
0
0
0
0
0
0
a 2D2
ꢃ
+2.3 (c 1.03, CHCl3). 1H NMR
(s, 3H, NHCOCH3), 1.67 (ddd, 1H, J3a ,2 = 11.8 Hz, J3a ,4 = 11.8 Hz,
H-3a0), 1.26 (s, 9H, (CH3)3CCO–). 13C NMR (125 MHz, CDCl3): d
177.6 ((CH3)3CCO–), 171.7 (NHCOCH3), 137.3 (Ph), 129.1 (Ph),
128.3 (Ph), 126.2 (Ph), 105.7 (C-10), 101.7 (PhCH), 101.1 (C-1),
75.9 (C-3), 72.9 (C-40), 71.3 (C-6), 70.7 (C-50), 69.4 (C-5), 69.3 (C-
4), 69.0 (C-60), 68.6 (C-20), 59.3 (C-6-OCH3), 56.7 (C-1-OCH3), 53.7
(C-2), 39.2 ((CH3)3CCO–), 35.1 (C-30), 27.3 ((CH3)3CCO–), 23.8
(NHCOCH3). Anal. Calcd for C28H41NO11(667.63): C, 59.25; H,
7.28; N, 2.47. Found: C, 59.13; H, 7.60; N, 2.46.
0
0
0
0
(1.7 g, 2.6 mmol, 85%):
½
(500 MHz, CDCl3): d 7.52–7.48 (m, 2H, PhH), 7.40–7.31 (m, 3H,
PhH), 5.66 (d, 1H, JNH,2 = 7.4 Hz, NHCOCH3), 5.51 (s, 1H, PhCH), 5.37
(d, 1H, J4,3 = 3.1 Hz, H-4), 4.69 (d, 1H, J1,2 = 8.2 Hz, H-1), 4.37 (d, 1H,
J1 ,2 = 7.5 Hz, H-10), 4.67 (dd, 1H, J3,2 = 10.6 Hz, H-3), 4.26 (dd, 1H,
0
0
J6e ,6a = 10.6 Hz, J6e ,5 = 4.9 Hz, H-6e0), 4.14 (dd, 1H, J6a,6b = 10.4 Hz,
J6a,5 = 6.0 Hz, H-6a), 4.10 (dd, 1H, J6b,5 = 7.3 Hz, H-6b), 3.92 (dd, 1H,
H-5), 3.76–3.65 (m, 1H, H-6a0, H-2), 3.57–3.53 (m, 1H, H-40), 3.51
0
0
0
0
(s, 3H, OCH3), 3.49 (ddd, 1H, J2 ,3a = 12.3 Hz, J2 ,3e = 4.8 Hz, H-20),
0
0
0
0
3.39 (ddd, 1H, J5 ,4 = 9.7, J5 ,6a = 9.7 Hz, H-50), 2.42 (ddd, 1H, J3e ,3a
=
0
0
0
0
0
0
11.8 Hz, J3e ,4 = 4.7 Hz, H-3e0), 2.02 (s, 3H, NHCOCH3), 1.68 (ddd,
3.20. Methyl 2-acetamido-3-O-(4,6-O-benzylidene-3-deoxy-b-D-
0
0
1H, J4 ,3a = 11.8 Hz, H-3a0), 1.25 (s, 9H, (CH3)3CCO–), 1.21 (s, 9H,
(CH3)3CCO–). 13C NMR (125 MHz, CDCl3): d 177.9 ((CH3)3CCO–),
177.2 ((CH3)3CCO–), 171.6 (NHCOCH3), 137.2 (Ph), 129.1 (Ph),
128.3 (Ph), 126.1 (Ph), 105.6 (C-10), 101.7 (PhCH), 100.9 (C-1), 75.8
(C-40), 75.5 (C-3), 71.3 (C-5), 70.6 (C-50), 68.9 (C-60), 68.7 (C-4),
68.6 (C-20), 62.0 (C-6), 56.7 (OCH3), 54.1 (C-2), 39.1 ((CH3)3CCO–),
38.7 ((CH3)3CCO–), 33.0 (C-30), 27.2 ((CH3)3CCO–), 27.0 ((CH3)3-
CCO–), 23.8 (NHCOCH3). Anal. Calcd for C32H47NO12(637.72): C,
60.27; H, 7.43; N, 2.20. Found: C, 59.86; H, 7.55; N, 2.21.
arabino-hexopyranosyl)- 2-deoxy-4,6-di-O-pivaloyl-b-
galactopyranoside (27)
D
-
0
0
After drying under vacuum for 18 h, the syrup of crude 25a was
dissolved in dry distilled THF (10 mL), and the reaction mixture
was cooled at 0 °C under argon. A solution of L-selectride in THF
(5.0 mL, 1.0 M) was added slowly by syringe, and the reaction
was quenched with methanol immediately when bubbling ceased.
Evaporation of the solvents and purification by column chromatog-
raphy (acetone/toluene, 1:5) afforded the compound 27 as a white
3.18. Methyl 2-acetamido-3-O-(4,6-O-benzylidene-3-deoxy-b-
erythro-hexos-2-ulosyl)-2- deoxy-4,6-di-O-pivaloyl-b-
galactopyranoside (25a)
D-
solid (0.99 g, 76% over 2 steps): ½a D22
ꢃ
ꢂ2.27 (c 22.0, CHCl3). 1H NMR
D
-
(500 MHz, CDCl3): d 7.49–7.45 (m, 2H, PhH), 7.39–7.32 (m, 3H,
PhH), 5.69 (d, 1H, JNH,2 = 7.1 Hz, NHCOCH3), 5.55 (s, 1H, PhCH),
5.38 (d, 1H, J4,3 = 3.2 Hz, H-4), 4.91 (d, 1H, J1,2 = 8.2 Hz, H-1), 4.86
(dd, 1H, J3,2 = 11.0 Hz, H-3), 4.68 (s, 1H, H-10), 4.26 (dd, 1H, J6e ,6a
=
0
0
Disaccharide 25 (1.30 g, 2.0 mmol) was dissolved in a mixture
of dry distilled dimethylsulfoxide (6.0 mL) and acetic anhydride
(3.0 mL) under argon, and the reaction stirred for 96 h. Evaporation
of the solvents gave a yellowish syrup 25a, which can be used di-
rectly in the next step. It was purified by column chromatography
10.6 Hz, J6e ,5 = 4.9 Hz, H-6e0), 4.16–4.08 (m, high order, 2H, H-6a,
0
0
0
0
0
0
0
0
H-6b), 3.99 (ddd, 1H, J4 ,3a = 11.5 Hz, J4 ,5 = 9.4 Hz, J4 ,3e = 4.5 Hz,
H-40), 3.96 (dd, 1H, J5,6a = 6.7 Hz, J5,6b = 6.7 Hz, H-5), 3.90 (app. s,
1H, H-20), 3.80 (dd, 1H, J6a ,5 = 10.3 Hz, H-6a0), 3.52 (s, 3H, OCH3),
0
0
(acetone/toluene, 1:5) for characterization purpose only: ½a D22
ꢃ
3.42 (ddd, 1H, H-50), 3.29 (ddd, 1H, H-2), 2.33 (ddd, 1H, J3e ,3a
=
0
0
+2.86 (c 2.1, CHCl3). 1H NMR (600 MHz, CDCl3): d 7.52–7.48 (m,
2H, PhH), 7.40–7.16 (m, 3H, PhH), 5.76 (d, 1H, JNH,2 = 6.9 Hz,
NHCOCH3), 5.66 (s, 1H, PhCH), 5.49 (d, 1H, J4,3 = 3.0 Hz, H-4), 5.00
(d, 1H, J1,2 = 8.3 Hz, H-1), 4.82 (s, 1H, H-10), 4.80 (dd, 1H,
13.3 Hz, J3e ,2 = 4.0 Hz, H-3e0), 2.00 (s, 3H, NHCOCH3), 1.70 (ddd,
0
0
1H, J3a ,2 = 3.0 Hz, H-3a0), 1.25 (s, 9H, (CH3)3CCO–), 1.20 (s, 9H,
(CH3)3CCO–). 13C NMR (125 MHz, CDCl3): d 178.1 ((CH3)3CCO–),
177.9 ((CH3)3CCO–), 171.0 (NHCOCH3), 137.5 (Ph), 129.0 (Ph),
128.3 (Ph), 126.2 (Ph), 102.0 (PhCH), 101.1 (C-10), 100.3 (C-1),
73.3 (C-3), 72.9 (C-40), 71.0 (C-5 or C-50), 70.8 (C-50 or C-5), 69.5
(C-4), 68.9 (C-60), 67.6 (C-20), 61.7 (C-6), 57.1 (OCH3), 55.5 (C-2),
39.3 ((CH3)3CCO–), 38.7 ((CH3)3CCO–), 34.1 (C-30), 27.2
((CH3)3CCO–), 27.1 ((CH3)3CCO–), 23.8 (NHCOCH3). ES HRMS calcd
for C32H47NO12Na (M+Na): 660.2991, found: 660.2993.
0
0
0
0
0
0
J3,2 = 10.8 Hz, H-3), 4.37 (dd, 1H, J6e ,6a = 10.6 Hz, J6e ,5 = 5.0 Hz, H-
6e0), 4.21 (ddd, 1H, J4 ,3a = 11.6 Hz, J4 ,5 = 9.7 Hz, J4 ,3e = 6.1 Hz, H-
0
0
0
0
0
0
40), 4.12–4.08 (m, high order, 2H, H-6a, H-6b), 3.92–3.98 (m, 2H,
H-6a0, H-5), 3.73 (ddd, 1H, J5 ,6a = 9.7 Hz, H-50), 3.52 (s, 3H, OCH3),
0
0
3.21 (ddd, 1H, H-2), 3.00 (dd, 1H, J3e ,3a = 17.1 Hz, H-3e0), 2.53
(dd, 1H, H-3a0), 2.02 (s, 3H, NHCOCH3), 1.25 (s, 9H, (CH3)3CCO–),
1.22 (s, 9H, (CH3)3CCO–). 13C NMR (100 MHz, CDCl3): d 197.8 (C-
20), 177.7 ((CH3)3CCO–), 177.0 ((CH3)3CCO–), 171.5 (NHCOCH3),
136.9 (Ph), 129.1 (Ph), 128.3 (Ph), 128.2 (Ph), 126.0 (Ph), 101.1
(PhCH), 100.5 (C-10), 100.1 (C-1), 74.4 (C-50), 73.2 (C-40), 71.0 (C-
5), 69.7 (C-3), 68.9 (C-60), 68.5 (C-4), 61.5 (C-6), 57.0 (OCH3), 55.8
(C-2), 42.5 (C-30), 39.0 ((CH3)3CCO–), 38.6 ((CH3)3CCO–), 27.1
((CH3)3CCO–), 27.0 ((CH3)3CCO–), 23.6 (NHCOCH3). ES HRMS calcd
for C32H45NO12Na (M+Na): 658.2834, found: 658.2838.
0
0
3.21. Methyl 2-acetamido-3-O-(4,6-O-benzylidene-3-deoxy-2-O-
methyl-b-D-arabino- hexopyranosyl)-2-deoxy-4,6-di-O-pivaloyl-
b-D
-galactopyranoside (28)
To a solution of compound 28 (104 mg, 0.16 mmol) and methyl
iodide (52
lL, 0.80 mmol) in dry DMF (1 mL) was added sodium
hydride (20 mg, 60% dispersion in mineral oil, 0.48 mmol). The

10
L. Cui et al. / Carbohydrate Research 383 (2014) 1–13
reaction mixture was stirred at rt. for 1 h, and water was added to
quench the reaction. The solution was evaporated to dryness, and
column chromatography (acetone/toluene, 1:5) gave the methyl-
the bromination, the solution turned orange, and the reaction
reached completion when the orange color disappeared. The reac-
tion mixture was applied directly to a column and chromato-
graphed (acetone/toluene, 1:5) to give compound 30 as a white
ated product (35 mg, 0.054 mmol, 33%): ½a D22
ꢃ
+7.7 (c 0.43, CHCl3).
1H NMR (500 MHz, CDCl3): d 7.48–7.42 (m, 2H, PhH), 7.37–7.30
(m, 3H, PhH), 5.66 (d, 1H, JNH,2 = 7.3 Hz, NHCOCH3), 5.52 (s, 1H,
PhCH), 5.43 (d, 1H, J4,3 = 2.8 Hz, H-4), 4.86 (d, 1H, J1,2 = 8.4 Hz, H-
solid (186 mg, 74%): ½a D22
ꢃ
+16.8 (c 3.5, CHCl3). 1H NMR (600 MHz,
CDCl3): d 8.01–8.00 (m, 2H, PhH), 7.60–7.58 (m, 1H, PhH), 7.47–
7.44 (m, 2H, PhH), 5.71 (d, 1H, JNH,2 = 7.5 Hz, NHCOCH3), 5.54 (d,
1), 4.68 (d, 1H, J1 ,2 = 1.4 Hz, H-10), 4.61 (dd, 1H, J3,2 = 10.9 Hz, H-
1H, J4,3 = 3.0 Hz, H-4), 5.25 (ddd, 1H, J4 ,3a = 10.2 Hz, J4 ,5 = 10.2 Hz,
0
0
0
0
0
0
3), 4.21 (dd, 1H, J6e ,6a = 10.5 Hz, J6e ,5 = 4.9 Hz, H-6e0), 4.10 (dd,
1H, J6a,6b = 11.4 Hz, J6a,5 = 6.1 Hz, H-6a), 4.04 (dd, 1H, J6b,5 = 7.3 Hz,
J4 ,3e = 4.9 Hz, H-40), 4.86 (dd, 1H, J3,2 = 11.1 Hz, H-3), 4.81 (d, 1H,
J1,2 = 8.6 Hz, H-1), 4.79 (s, 1H, H-10), 4.20 (dd, 1H, J6a,6b = 11.1 Hz,
J6a,5 = 6.9 Hz, H-6a), 4.10 (dd, 1H, J6b,5 = 7.1 Hz, H-6b), 4.00 (dd, 1H,
0
0
0
0
0
0
H-6b), 3.92–3.84 (m, 2H, H-40, H-5), 3.79 (dd, 1H, J6a ,5 = 10.3 Hz,
0
0
H-6a0), 3.58 (ddd, 1H, J2 ,3a = 4.1 Hz, J2 ,3e = 4.1 Hz, H-20), 3.49 (s,
H-5), 3.87 (app. s, 1H, H-20), 3.82 (ddd, 1H, J5 ,6a = 9.1 Hz, J5 ,6b
=
0
0
0
0
0
0
0
0
3H, C-1-OCH3), 3.48–3.42 (m, 1H, H-50), 3.41 (s, 3H, C-6-OCH3),
2.6 Hz, H-50), 3.58 (dd, 1H, J6b ,6a = 11.1 Hz, H-6b0), 3.55–3.50 (m,
0
0
3.38–3.31 (m, 1H, H-2), 2.28 (ddd, 1H, J3e ,3a = 12.3 Hz, J3e ,4 = 4.8 -
2H, H-2, H-6a0), 3.53 (s, 3H, OCH3), 2.50 (ddd, 1H, J3e ,3a = 13.6 Hz,
0
0
0
0
0
0
Hz, H-3e0), 1.97 (s, 3H, NHCOCH3), 1.71 (ddd, 1H, J3a ,4 = 11.1 Hz, H-
3a0), 1.23 (s, 9H, (CH3)3CCO–), 1.18 (s, 9H, (CH3)3CCO–). 13C NMR
(100 MHz, CDCl3): d 177.8 ((CH3)3CCO–), 177.0 ((CH3)3CCO–),
170.7 (NHCOCH3), 137.4 (Ph), 128.9 (Ph), 128.2 (Ph), 126.0 (Ph),
101.6 (PhCH), 101.4 (C-10), 100.6 (C-1), 76.1 (C-20), 74.0, 73.1,
71.3, 70.5 (C-4), 68.9 (C-60), 68.4, 62.0 (C-6), 57.9 (C-20-OCH3),
56.8 (C-1-OCH3), 55.1 (C-2), 39.3 ((CH3)3CCO–), 38.7 ((CH3)3CCO–),
31.3 (C-30), 27.2 ((CH3)3CCO–), 27.0 ((CH3)3CCO–), 23.7 (NHCOCH3).
ES HRMS calcd for C33H49NO12Na (M+Na): 674.3147, found:
674.3142.
J3e ,2 = 4.5 Hz, H-3e0), 2.03 (s, 3H, NHCOCH3), 1.70 (ddd, 1H,
0
0
0
0
J3a ,2 = 2.7 Hz, H-3a0), 1.26 (s, 9H, (CH3)3CCO–), 1.21 (s, 9H, (CH3)3
CCO–). 13C NMR (125 MHz, CDCl3): d 178.4 ((CH3)3CCO–), 177.9
((CH3)3CCO–), 171.0 (NHCOCH3), 165.2 (PhCO–), 133.4 (Ph), 129.7
(Ph), 129.5 (Ph), 128.5 (Ph), 100.9 (C-1), 100.5 (C-10), 72.9 (C-40),
70.9 (C-5), 69.4 (C-4), 67.9 (C-3), 66.7 (C-20), 61.4 (C-6), 57.0
(OCH3), 54.7 (C-2), 39.4 ((CH3)3CCO–), 38.8 ((CH3)3CCO–), 34.1
(C-30), 32.3 (C-60), 27.2 ((CH3)3CCO–), 27.1 ((CH3)3CCO–), 23.8
(NHCOCH3). ES HRMS calcd for C32H46NO12BrNa (M+Na):
738.2096, found: 738.2093.
0
0
3.22. Methyl 2-acetamido-3-O-(4,6-O-benzylidene-3-deoxy-b-
D
-
3.24. Methyl 2-acetamido-3-O-(4-O-benzoyl-6-bromo-3,6-
arabino-hexopyranosyl)- 2-deoxy-6-O-methyl-4-O-pivaloyl-b-
D
-
dideoxy-2-O-methyl-b-
D- arabino-hexopyranosyl)-2-deoxy-4,6-
galactopyranoside (29)
di-O-pivaloyl-b- -galactopyranoside (31)
D
Compound 26 (0.18 g, 0.31 mmol) was dissolved in a mixture of
dry distilled dimethylsulfoxide (1.4 mL) and acetic anhydride
(0.7 mL) under argon, and the reaction was stirred for 48 h. Evapora-
tion of the solvents gave a yellowish syrup, which was dissolved in
dry distilled THF (2 mL), and the reaction mixture was cooled at
Compound 28 (35 mg, 0.054 mmol) was dissolved in carbon tet-
rachloride (1.0 mL), followed by addition of N-bromosuccinimide
(18 mg, 0.081 mmol) and barium carbonate (24 mg, 0.70 mmol).
The reaction mixture was refluxed under argon for 70 min, cooled
to ambient temperature and applied directly to a column and chro-
matographed (acetone/toluene, 1:5) to produce compound 31 as a
ꢂ20 °C under argon. A solution of
L-selectride in THF (0.8 mL,
1.0 M) was added slowly by syringe, and the reaction was quenched
with methanol after 1 min. Evaporation of the solvents and purifica-
tion by column chromatography (acetone/toluene, 1:2) afforded
white solid (27 mg, 72%): ½a D22
ꢃ
+11.0 (c 5.5, CHCl3). 1H NMR
(500 MHz, CDCl3): d 8.01–8.00 (m, 2H, PhH), 7.61–7.58 (m, 1H,
PhH), 7.48–7.46 (m, 2H, PhH), 5.80 (d, 1H, JNH,2 = 6.8 Hz, NHCOCH3),
compound 29 as a white solid (0.15 g, 86% over 2 steps): ½a D22
ꢃ
5.57 (d, 1H, J4,3 = 2.9 Hz, H-4), 5.17 (ddd, 1H, J4 ,3a = 9.4 Hz, J4 ,5 =
0
0
0
0
+0.92 (c 6.4, CHCl3). 1H NMR (600 MHz, CDCl3): d 7.50–7.45 (m,
2H, PhH), 7.40–7.32 (m, 3H, PhH), 5.66 (d, 1H, JNH,2 = 7.4 Hz,
NHCOCH3), 5.54 (s, 1H, PhCH), 5.38 (d, 1H, J4,3 = 3.2 Hz, H-4), 4.86
(d, 1H, J1,2 = 8.4 Hz, H-1), 4.77 (dd, 1H, J3,2 = 11.0 Hz, H-3), 4.67 (s,
9.4 Hz, J4 ,3e = 4.6 Hz, H-40), 4.82 (s, 1H, H-10), 4.65 (d, 1H,
J1,2 = 8.3 Hz, H-1), 4.60 (dd, 1H, J3,2 = 11.0 Hz, H-3), 4.13 (dd, 1H,
J6a,6b = 11.5 Hz, J6a,5 = 7.3 Hz, H-6a), 4.05 (dd, 1H, J6b,5 = 6.2 Hz, H-
6b), 3.92 (dd, 1H, H-5), 3.91–3.88 (m, 1H, H-50), 3.72 (ddd, 1H, H-
2), 3.62–3.59 (m, 1H, H-20), 3.58–3.54 (m, 1H, H-6a0, H-6b0), 3.51
0
0
1H, H-10), 4.28 (dd, 1H, J6e ,6a = 10.6 Hz, J6e ,5 = 5.0 Hz, H-6e0), 3.98
0
0
0
0
(ddd, 1H, J4 ,3a = 11.6 Hz, J4 ,5 = 9.5 Hz, J4 ,3e = 4.5 Hz, H-40), 3.90
(app. s, 1H, H-20), 3.85 (dd, 1H, J5,6a = 6.5 Hz, J5,6b = 6.5 Hz, H-5),
(s, 3H, C-1-OCH3), 3.46 (s, 3H, C-20-OCH3), 2.56 (ddd, 1H, J3e ,3a
=
0
0
0
0
0
0
0
0
13.6 Hz, J3e ,2 = 4.9 Hz, H-3e0), 2.04 (s, 3H, NHCOCH3), 1.67 (ddd,
0
0
3.82 (dd, 1H, J6a ,5 = 10.4 Hz, H-6a0), 3.54 (s, 3H, C-1-OCH3), 3.48
(dd, 1H, J6a,6b = 10.1 Hz, H-6a), 3.44 (dd, 1H, H-6b), 3.44–3.36 (m,
1H, J3a ,2 = 2.6 Hz, H-3a0), 1.26 (s, 9H, (CH3)3CCO–), 1.21 (s, 9H,
(CH3)3CCO–). 13C NMR (125 MHz, CDCl3): d 178.0 ((CH3)3CCO–),
177.3 ((CH3)3CCO–), 170.8 (PhCO–), 165.3 (NHCOCH3), 133.5 (Ph),
129.6 (Ph), 129.5 (Ph), 128.5 (Ph), 101.7 (C-1), 100.0 (C-10), 77.0
(C-50), 75.5 (C-20), 73.5 (C-3), 71.4 (C-5), 68.3 (C-4), 67.9 (C-40),
62.1 (C-6), 57.5 (C-20-OCH3), 56.7 (C-1-OCH3), 53.9 (C-2), 39.1
((CH3)3CCO–), 38.7 ((CH3)3CCO–), 32.3 (C-60), 30.3 (C-30), 27.3
((CH3)3CCO–), 27.1 ((CH3)3CCO–), 23.9 (NHCOCH3). ES HRMS calcd
for C33H48NO12BrNa (M+Na): 752.2252, found: 752.2251.
0
0
0
0
2H, H-50, H-2), 3.35 (s, 3H, C-6-OCH3), 2.41 (ddd, 1H, J3e ,3a = 13.3 Hz,
0
0
J3e ,2 = 4.0 Hz, H-3e0), 2.00 (s, 3H, vNHCOCH3), 1.73–1.68 (m, 1H, H-
3a0), 1.26 (s, 9H, (CH3)3CCO–). 13C NMR (125 MHz, CDCl3): d 177.9
((CH3)3CCO–), 171.0 (NHCOCH3), 137.5 (Ph), 129.0 (Ph), 128.3 (Ph),
126.2 (Ph), 101.9 (PhCH), 100.9 (C-10), 100.5 (C-1), 73.3 (C-40), 73.2
(C-3), 72.6 (C-5), 71.2 (C-6), 70.8 (C-50), 70.0 (C-4), 69.0 (C-60), 67.6
(C-20), 59.3 (C-6-OCH3), 57.0 (C-1-OCH3), 55.2 (C-2), 39.1
((CH3)3CCO–), 34.0 (C-30), 27.2 ((CH3)3CCO–), 23.7 (NHCOCH3). ES
HRMS calcd for C28H41NO11Na (M+Na): 590.2572, found: 590.2570.
0
0
3.25. Methyl 2-acetamido-3-O-(4-O-benzoyl-6-bromo-3,6-
dideoxy-b-
D
-arabino-hexopyranosyl)-2-deoxy-6-O-methyl-4-O-
3.23. Methyl 2-acetamido-3-O-(4-O-benzoyl-6-bromo-3,6-
pivaloyl-b-
D-galactopyranoside (32)
dideoxy-b-
D-arabino- hexopyranosyl)-2-deoxy-4,6-di-O-
pivaloyl-b-
D
-galactopyranoside (30)
To a solution of 29 (75 mg, 0.13 mmol) in carbon tetrachloride
(1 mL), N-bromosuccinimide (44 mg, 0.20 mmol) and barium car-
bonate (56 mg, 0.26 mmol) were added. The reaction mixture
was kept at reflux under argon for 1 h, and then cooled. During
the bromination, the solution turned orange, and the reaction
reached completion when the orange color disappeared. The crude
To a solution of 27 (222 mg, 0.35 mmol) in carbon tetrachloride
(2.0 mL), N-bromosuccinimide (119 mg, 0.53 mmol) and barium
carbonate (151 mg, 0.70 mmol) were added. The reaction mixture
was heated at reflux under argon for 1 h, and then cooled. During

L. Cui et al. / Carbohydrate Research 383 (2014) 1–13
11
mixture was applied directly to a column and chromatographed
4.72 (d, 1H, J1,2 = 8.5 Hz, H-1), 4.69 (s, 1H, H-10), 4.60 (dd, 1H,
J3,2 = 11.0 Hz, H-3), 4.14 (dd, 1H, J6a,6b = 11.2 Hz, J6a,5 = 6.2 Hz, H-
6a), 4.09 (dd, 1H, J6b,5 = 7.2 Hz, H-6b), 3.91 (dd, 1H, H-5), 3.78
(acetone/toluene, 1:5) to produce compound 32 as a white solid
(69 mg, 82%): ½a 2D2
ꢃ
+5.0 (c 11.5, CHCl3). 1H NMR (500 MHz, CDCl3):
d 8.01–7.99 (m, 2H, PhH), 7.61–7.57 (m, 1H, PhH), 7.46–7.42 (m,
(dq, 1H, J5 ,6 = 6.2 Hz, H-50), 3.62 (app. s, 1H, H-20), 3.55–3.50 (m,
0
0
2H, PhH), 5.68 (d, 1H, JNH,2 = 7.9 Hz, NHCOCH3), 5.53 (d, 1H,
1H, H-2), 3.51 (s, 3H, C-1-OCH3), 3.47 (s, 3H, C-20-OCH3), 2.41
J4,3 = 2.7 Hz, H-4), 5.25 (ddd, 1H, J4 ,3a = 10.3 Hz, J4 ,5 = 10.3 Hz,
(ddd, 1H, J3e ,3a = 13.8 Hz, J3e ,2 = 4.4 Hz, H-3e0), 2.00 (s, 3H,
0
0
0
0
0
0
0
0
J4 ,3e = 4.9 Hz, H-40), 4.78 (s, 1H, H-10), 4.77 (dd, 1H, J3,2 = 10.8 Hz,
NHCOCH3), 1.56 (ddd, 1H, J3a ,2 = 2.4 Hz, H-3a0), 1.31 (d, 3H, H-60),
1.25 (s, 9H, (CH3)3CCO–), 1.20 (s, 9H, (CH3)3CCO–). 13C NMR
(125 MHz, CDCl3): d 177.9 ((CH3)3CCO–), 177.4 ((CH3)3CCO–),
170.8 (NHCOCH3), 165.6 (PhCO–), 133.1 (Ph), 130.0 (Ph), 129.6
(Ph), 128.4 (Ph), 101.5 (C-1), 99.9 (C-10), 76.0 (C-20), 74.5 (C-50),
73.0, 71.4, 70.4, 67.8 (C-4), 62.3 (C-6), 57.0 (C-20-OCH3), 56.8 (C-
1-OCH3), 54.2 (C-2), 39.1 ((CH3)3CCO–), 38.7 ((CH3)3CCO–), 30.5
(C-30), 27.3 ((CH3)3CCO–), 27.1 ((CH3)3CCO–), 23.8 (NHCOCH3),
18.4 (C-60). ES HRMS calcd for C33H49NO12Na (M+Na): 674.3147,
found: 674.3145.
0
0
0
0
H-3), 4.74 (d, 1H, J1,2 = 8.3 Hz, H-1), 3.89–3.84 (m, 2H, H-5, H-20),
3.81 (ddd, 1H, J5 ,6a = 8.4 Hz, J5 ,6b = 8.4 Hz, H-50), 3.62–3.51 (m, 3H,
0
0
0
0
H-2, H-6a0, H-6b0), 3.54 (s, 3H, C-1-OCH3), 3.51–3.44 (m, 2H, H-6a,
0
0
H-6b), 3.36 (s, 3H, C-6-OCH3), 2.49 (ddd, 1H, J3e ,3a = 13.7 Hz,
0
0
0
0
0
J3e ,2 = 4.4 Hz, H-3e ), 2.03 (s, 3H, NHCOCH3), 1.70 (ddd, 1H, J3a ,2 = 2.9 -
Hz, H-3a0), 1.26 (s, 9H, (CH3)3CCO–). 13C NMR (125 MHz, CDCl3): d
178.4 ((CH3)3CCO–), 170.8 (NHCOCH3), 165.2 (PhCO–), 133.4 (Ph),
129.7 (Ph), 129.5 (Ph), 128.5 (Ph), 101.0 (C-10), 100.4 (C-1), 77.4
(C-50), 73.2 (C-3), 72.6 (C-5), 71.3 (C-6), 70.1 (C-4), 67.9 (C-40), 66.7
(C-20), 59.3 (C-6-OCH3), 56.9 (C-1-OCH3), 54.5 (C-2), 39.1 ((CH3)3
CCO–), 33.9 (C-30), 32.4 (C-60), 27.2 ((CH3)3CCO–), 23.9 (NHCOCH3).
ES HRMS calcd for C28H40BrNO11Na (M+Na): 668.1677, found:
668.1680.
3.28. Methyl 2-acetamido-3-O-(4-O-benzoyl-3,6-dideoxy-b-D-
arabino-hexopyranosyl)- 2-deoxy-6-O-methyl-4-O-pivaloyl-b-D-
galactopyranoside (35)
3.26. Methyl 2-acetamido-3-O-(4-O-benzoyl-3,6-dideoxy-b-
D-
Compound 32 (36 mg, 0.056 mmol) was dissolved in dry tolu-
ene (1 mL). After tributyltin hydride (33 L, 0.067 mmol) and azob-
arabino-hexopyranosyl)- 2-deoxy-4,6-di-O-pivaloyl-b-
D
-
l
galactopyranoside (33)
isisobutyronitrile (AIBN) (catalytic amount) were added, the
reaction mixture was refluxed for 1 h. The reaction mixture was
cooled, applied directly to a column and chromatographed (ace-
tone/toluene, 1:4) to give compound 35 as a white solid (29 mg,
Compound 30 (35 mg, 0.049 mmol) was dissolved in dry tolu-
ene (1 mL). After tributyltin hydride (25 L, 0.059 mmol) and azob-
l
isisobutyronitrile (AIBN) (catalytic amount) were added, the
reaction mixture was refluxed for 1 h. The reaction mixture was
cooled, applied directly to a column and chromatographed (ace-
tone/toluene, 1:4) to give the 3,6-dideoxy sugar 33 as a white solid
91%): ½a 2D2
ꢃ
+11.8 (c 12.0, CHCl3). 1H NMR (400 MHz, CDCl3): d
8.00–7.96 (m, 2H, PhH), 7.59–7.50 (m, 1H, PhH), 7.43–7.37 (m,
2H, PhH), 5.65 (d, 1H, JNH,2 = 7.0 Hz, NHCOCH3), 5.41 (d, 1H,
0
0
0
0
J4,3 = 3.1 Hz, H-4), 5.06 (ddd, 1H, J4 ,3a = 10.7 Hz, J4 ,5 = 10.7 Hz,
(32 mg, 100%): ½a D22
ꢃ
+15.5 (c 2.5, CHCl3). 1H NMR (600 MHz, CDCl3):
d 8.01–7.99 (m, 2H, PhH), 7.60–7.55 (m, 1H, PhH), 7.45–7.42 (m,
J4 ,3e = 4.6 Hz, H-40), 4.90 (d, 1H, J1,2 = 8.4 Hz, H-1), 4.80 (dd, 1H,
0
0
J3,2 = 11.1 Hz, H-3), 4.65 (s, 1H, H-10), 3.86–3.84 (m, 2H, H-5, H-
20), 3.65 (dq, 1H, J5 ,6 = 6.2 Hz, H-50), 3.52 (s, 3H, C-1-OCH3), 3.47–
0
0
2H, PhH), 5.71 (d, 1H, JNH,2 = 7.1 Hz, NHCOCH3), 5.45 (d, 1H,
0
0
0
0
J4,3 = 3.3 Hz, H-4), 5.09 (ddd, 1H, J4 ,3a = 10.6 Hz, J4 ,5 = 10.6 Hz,
3.43 (m, 2H, H-6a, H-6b), 3.32 (s, 3H, C-6-OCH3), 3.32–3.28 (m,
J4 ,3e = 4.9 Hz, H-40), 4.96 (d, 1H, J1,2 = 8.3 Hz, H-1), 4.89 (dd, 1H,
J3,2 = 11.0 Hz, H-3), 4.68 (s, 1H, H-10), 4.15 (dd, 1H, J6a,6b = 11.2 Hz,
J6a,5 = 7.5 Hz, H-6a), 4.11 (dd, 1H, J6b,5 = 7.5 Hz, H-6b), 3.97 (dd,
1H, H-2), 2.45 (ddd, 1H, J3e ,3a = 13.7 Hz, J3e ,2 = 4.3 Hz, H-3e0),
1.99 (s, 3H, NHCOCH3), 1.62–1.55 (m, 1H, H-3a0), 1.27 (d, 3H, H-
60), 1.23 (s, 9H, (CH3)3CCO–). 13C NMR (100 MHz, CDCl3): d 177.9
((CH3)3CCO–), 170.8 (NHCOCH3), 165.2 (PhCO–), 132.9 (Ph), 130.0
(Ph), 129.4 (Ph), 128.2 (Ph), 100.5 (C-10), 100.3 (C-1), 73.8 (C-50),
72.7 (C-5), 72.6 (C-3), 71.2 (C-6), 70.0 (C-4), 69.8 (C-40), 67.1 (C-
20), 59.2 (C-6-OCH3), 57.0 (C-1-OCH3), 55.4 (C-2), 39.4
((CH3)3CCO–), 34.1 (C-30), 27.1 ((CH3)3CCO–), 23.7 (NHCOCH3),
18.0 (C-60). ES HRMS calcd for C28H41NO11Na (M+Na): 590.2572,
found: 590.2573.
0
0
0
0
0
0
1H, H-5), 3.86 (app. d, 1H, J2 ,OH = 2.2 Hz, H-20), 3.68 (dq, 1H,
0
J5 ,6 = 6.2 Hz, H-50), 3.52 (s, 3H, OCH3), 3.25 (ddd, 1H, H-2), 2.56
0
0
(d, 1H, C-20-OH), 2.48 (ddd, 1H, J3e ,3a = 13.5 Hz, J3e ,2 = 4.4 Hz,
H-3e0), 2.01 (s, 3H, NHCOCH3), 1.63 (m, 1H, H-3a0), 1.28 (d, 1H,
C-60), 1.25 (s, 9H, (CH3)3CCO–), 1.20 (s, 9H, (CH3)3CCO–). 13C NMR
(125 MHz, CDCl3): d 178.0 ((CH3)3CCO–), 177.9 ((CH3)3CCO–),
171.0 (NHCOCH3), 165.4 (PhCO–), 133.0 (Ph), 130.1 (Ph), 129.6
(Ph), 128.4 (Ph), 100.8 (C-10), 100.4 (C-1), 73.9 (C-50), 72.6 (C-3),
71.1 (C-5), 70.1 (C-40), 69.3 (C-4), 67.2 (C-20), 61.7 (C-6), 57.1
(OCH3), 55.6 (C-2), 39.3 ((CH3)3CCO–), 38.7 ((CH3)3CCO–), 34.4
(C-30), 27.2 ((CH3)3CCO–), 27.1 ((CH3)3CCO–), 23.8 (NHCOCH3),
18.0 (C-60). ES HRMS calcd for C32H47NO12Na (M+Na): 660.2991,
found: 660.2991.
0
0
0
0
3.29. Methyl 2-acetamido-3-O-(3,6-dideoxy-b-
D-arabino-
hexopyranosyl)-2-deoxy- b- -galactopyranoside (2)
D
Compound 33 (26 mg, 0.041 mmol) was dissolved in dry dis-
tilled methanol (1 mL) under argon. Sodium methoxide in metha-
nol solution (1.7 M, 29 lL) was added, and the reaction mixture
3.27. Methyl 2-acetamido-3-O-(4-O-benzoyl-3,6-dideoxy-2-O-
methyl-b-D-arabino-hexopyranosyl)- 2-deoxy-4,6-di-O-pivaloyl-
was stirred at rt. for 22 h. Dowex 50WX4-50 H+ resin was added
to neutralize the solution, and the resin was removed by filtration.
The filtrate was evaporated to dryness and the residue was purified
by HPLC using a Beckman C18 silica column. MeOH–H2O gradient
b-
D-galactopyranoside (34)
Compound 31 (19 mg, 0.026 mmol) was dissolved in dry tolu-
elution afforded the native disaccharide 2 (15 mg, 100%): ½a D22
ꢃ
ene (1 mL). Tributyltin hydride (15
lL, 0.032 mmol) and azobisi-
ꢂ27.5 (c 0.50, CH3OH). 1H NMR (600 MHz, CD3OD): d 4.55 (d, 1H,
J1 ,2 = 1.0 Hz, H-10), 4.34 (d, 1H, J1,2 = 8.4 Hz, H-1), 4.04 (d, 1H,
J4,3 = 2.5 Hz, H-4), 4.01 (dd, 1H, J2,3 = 10.7 Hz, H-2), 3.78–3.72 (m,
3H, H-20, H-3, H-6a, H-6b), 3.52–3.46 (m, 2H, H-5, H-40), 3.45 (s,
0
0
sobutyronitrile (AIBN) (catalytic amount) were added, the
reaction mixture was refluxed for 50 min, cooled, applied directly
to a column and chromatographed (acetone/toluene, 1:5) to give
compound 34 as a white solid (15 mg, 0.023 mmol, 87%): ½a D22
ꢃ
3H, OCH3), 3.26 (dd, 1H, J5 ,4 = 9.2 Hz, J5 ,6 = 6.1 Hz, H-50), 2.12
0
0
0
0
+19.0 (c 4.5, CHCl3). 1H NMR (600 MHz, CDCl3): d 8.01–8.00 (m,
2H, PhH), 7.60–7.56 (m, 1H, PhH), 7.44–7.40 (m, 2H, PhH), 5.78
(d, 1H, JNH,2 = 6.8 Hz, NHCOCH3), 5.48 (d, 1H, J4,3 = 3.2 Hz, H-4),
(ddd, 1H, J3e ,3a = 13.7 Hz, J3e ,4 = 4.7 Hz, J3e ,2 = 3.3 Hz, H-3e0), 1.94
0
0
0
0
0
0
0
0
0
0
(s, 3H, NHCOCH3), 1.51 (ddd, 1H, J3a ,4 = 11.4 Hz, J3a ,2 = 2.7 Hz, H-
3a0), 1.26 (d, 3H, H-60). 13C NMR (125 MHz, CD3OD): d 173.7
(NHCOCH3), 104.1 (C-10), 103.6 (C-1), 81.0 (C-3), 77.5 (C-40), 76.3
4.93 (ddd, 1H, J4 ,3a = 10.0 Hz, J4 ,5 = 10.4 Hz, J4 ,3e = 4.8 Hz, H-40),
0
0
0
0
0
0

12
L. Cui et al. / Carbohydrate Research 383 (2014) 1–13
(C-5), 69.8 (C-4), 69.2 (C-20), 68.1 (C-50), 62.4 (C-6), 56.9 (OCH3),
52.9 (C-2), 38.9 (C-30), 23.0 (NHCOCH3), 18.4 (C-60). ES HRMS calcd
for C15H27NO9Na (M+Na): 388.1578, found: 388.1576.
½
a 2D2
ꢃ
ꢂ26.3 (c 0.32, CH3OH). 1H NMR (600 MHz, D2O): d 4.78 (s, 1H,
H-10), 4.24 (d, 1H, J1,2 = 8.1 Hz, H-1), 4.10 (app. s, 1H, H-4), 4.08
(app. s, 1H, H-20), 3.80 (dd, 1H, J6a,6b = 11.4 Hz, J6a,5 = 8.0 Hz, H-
6a), 3.75 (dd, 1H, J6b,5 = 4.1 Hz, H-6b), 3.68 (dd, 1H, H-5), 3.65
(dd, 1H, J3,2 = 10.7 Hz, J3,4 = 3.0 Hz, H-3), 3.56 (s, 3H, OCH3), 3.58–
3.52 (m, 1H, H-40), 3.49–3.43 (m, 1H, H-50), 2.91 (dd, 1H, H-2),
3.30. Methyl 2-acetamido-3-O-(3,6-dideoxy-2-O-methyl-b-
D-
arabino-hexopyranosyl)- 2-deoxy- b- -galactopyranoside (5)
D
2.18 (ddd, 1H, J3e ,3a = 13.8 Hz, J3e ,2 = 4.1 Hz, J3e ,4 = 4.1 Hz, H-3e0),
0
0
0
0
0
0
1.68 (ddd, 1H, J3a ,4 = 11.4 Hz, J3a ,2 = 2.6 Hz, H-3a0), 1.27 (d, J6 ,5
=
0
0
0
0
0
0
Compound 34 (15 mg, 0.023 mmol) was suspended in dry dis-
tilled methanol (1 mL) under argon. A methanolic solution of so-
6.2 Hz, H-60). 13C NMR (125 MHz, CD3OD): d 175.6 (NHCOCH3),
104.8 (C-1), 103.8 (C-10), 82.9 (C-5), 77.7 (C-50), 76.4 (C-40), 69.1
(C-4), 68.8 (C-20), 68.0 (C-3), 62.4 (C-6), 57.3 (OCH3), 53.6 (C-2),
39.0 (C-30), 18.4 (C-60). ES HRMS calcd for C13H25NO8Na (M+Na):
346.1472, found: 346.1472.
dium methoxide (1.7 M, 29 lL) was added, and the reaction
mixture was stirred at rt for 6 h. Dowex 50WX4-50 H+ resin was
added to neutralize the solution, and the resin was removed by fil-
tration. The filtrate was evaporated to dryness and the residue was
purified by HPLC using a Beckman C18 silica column. Gradient elu-
tion with MeOH–H2O afforded the final disaccharide 5 (8.7 mg,
3.33. Methyl 2-amino-3-O-(3,6-dideoxy-b-
hexopyranosyl)-2-deoxy-6-O-methyl- b- -galactopyranoside (6)
D-arabino-
0.017 mmol, 100%):
½
a 2D2
ꢃ
ꢂ23.7 (c 0.50, CH3OH). 1H NMR
D
(600 MHz, D2O): d 4.73 (s, 1H, H-10), 4.43 (d, 1H, J1,2 = 8.6 Hz, H-
1), 4.12 (d, 1H, J4,3 = 3.1 Hz, H-4), 4.01 (dd, 1H, J2,3 = 10.7 Hz, H-2),
3.82 (dd, 1H, J6a,6b = 11.8 Hz, J6a,5 = 8.0 Hz, H-6a), 3.80 (dd, 1H, H-
3), 3.76 (dd, 1H, J6b,5 = 4.3 Hz, H-6b), 3.69 (dd, 1H, H-5), 3.57 (dd,
Compound 4 (4.0 mg, 0.011 mmol) was dissolved in an etha-
nolic solution of potassium hydroxide (1 mL, 4 N) and the solution
was refluxed for 64 h. The reaction was stopped by evaporating the
ethanol, and the crude mixture was applied to a Sep-Pak cartridge,
followed by HPLC with a C18 silica column eluted by methanol–
water to afford amino sugar 6 as a white solid (3.5 mg, 100% after
1H, J2 ,3a = 3.1 Hz, J2 ,3e = 3.1 Hz, H-20), 3.51 (s, 3H, C-1-OCH3),
0
0
0
0
3.47–3.42 (m, 2H, H-40, H-50), 3.41 (s, 3H, C-20-OCH3), 2.36 (ddd,
1H, J3e ,3a = 13.9 Hz, J3e ,4 = 3.6 Hz, H-3e0), 2.02 (s, 3H, NHCOCH3),
0
0
0
0
1.53 (ddd, 1H, J3a ,4 = 11.0 Hz, H-3a0), 1.25 (d, 3H, J6 ,5 = 5.2 Hz, H-
recovery of s.m.): ½a D22
ꢃ
ꢂ20.0 (c 0.10, CH3OH). 1H NMR (600 MHz,
0
0
0
0
13
60). C NMR (125 MHz, D2O): d 175.3 (NHCOCH3), 103.9 (C-10),
D2O): d 4.78 (s, 1H, H-10), 4.24 (d, 1H, J1,2 = 8.1 Hz, H-1), 4.09
(app. s, 1H, H-4), 4.09 (app. s, 1H, H-20), 3.82 (dd, 1H,
J5,6a = 7.8 Hz, J5,6b = 4.1 Hz, H-5), 3.69 (dd, 1H, J6a,6b = 11.0 Hz, H-
6a), 3.66 (dd, 1H, H-6b), 3.66 (dd, 1H, J3,2 = 10.8 Hz, J3,4 = 3.1 Hz,
H-3), 3.59–3.54 (m, 1H, H-40), 3.56 (s, 3H, C-1-OCH3), 3.46 (dq,
102.9 (C-1), 81.0 (C-3), 77.9 (C-20), 77.0 (C-50), 75.7 (C-5), 68.7 (C-
4), 67.8 (C-40), 61.9 (C-6), 58.3 (C-20-OCH3), 57.8 (C-1-OCH3), 52.0
(C-2), 34.6 (C-30), 23.0 (NHCOCH3), 18.0 (C-60). ES HRMS calcd for
C16H29NO9Na (M+Na): 402.1735, found: 402.1733.
1H, J5 ,4 = 9.5 Hz, J5 ,6 = 6.1 Hz, H-50), 3.41 (s, 3H, C-6-OCH3), 2.92
0
0
0
0
0
0
0
0
0
0
3.31. Methyl 2-acetamido-3-O-(3,6-dideoxy-b-
D
-arabino-
(dd, 1H, H-2), 2.18 (ddd, 1H, J3e ,3a = 14.0 Hz, J3e ,2 = 4.1 Hz, J3e ,4 =
0
0
0
0
hexopyranosyl)-2-deoxy- 6-O-methyl-b- -galactopyranoside (4)
D
4.1 Hz, H-3e0), 1.68 (ddd, 1H, J3a ,4 = 11.8 Hz, J3a ,2 = 2.8 Hz, H-3a0),
1.27 (d, 3H, H-60). 13C NMR (125 MHz, CDCl3): d 106.1 (C-1),
104.0 (C-10), 83.9 (C-3), 77.6 (C-50), 74.7 (C-5), 73.1 (C-6), 69.4,
68.8, 68.1, 59.4 (C-6-OCH3), 57.3 (C-1-OCH3), 53.5 (C-2), 39.0 (C-
30), 18.4 (C-60). ES HRMS calcd for C14H27NO8Na (M+Na):
360.1629, found: 360.1625.
Compound 35 (15 mg, 0.026 mmol) was dissolved in dry dis-
tilled methanol (1 mL) under argon. Sodium methoxide in metha-
nol solution (1.7 N, 29 lL) was added, and the reaction mixture
was stirred at rt for 6 h. Dowex 50WX4-50 H+ resin was added to
neutralize the solution, and the resin was removed by filtration.
The filtrate was evaporated to dryness and the residue was purified
by HPLC using a Beckman C18 silica column with a MeOH-H2O gra-
3.34. Methyl 2-acetamido-3-O-(3,6-dideoxy-2-O-methyl-b-D-
arabino-hexopyranosyl) -2-deoxy- 6-O-methyl-b-D-
dient to afford the final compound 4 (10 mg, 100%): ½a D22
ꢃ
ꢂ21.0
galactopyranoside (7)
1
(c 0.30, CH3OH). H NMR (600 MHz, D2O): d 4.68 (s, 1H, H-10),
4.44 (d, 1H, J1,2 = 8.6 Hz, H-1), 4.09 (d, 1H, J4,3 = 1.1 Hz, H-4), 4.01
A milky suspension of compound 5 (8 mg, 0.020 mmol) and
dibutyltin oxide (6 mg, 0.024 mmol) in dry distilled methanol
(2 mL) was refluxed for 2 h until the reaction mixture became
clear. The methanol was then evaporated, the syrup was co-evap-
orated with toluene, until a dry white residue was obtained. After
drying the white solid under vacuo, it was dissolved in dry DMF
(1 mL). Cesium fluoride salt (9.3 mg, 0.060 mmol) and iodometh-
0
0
0
0
(dd, 1H, J2,3 = 10.6 Hz, H-2), 3.89 (dd, 1H, J2 ,3a = 3.1 Hz, J2 ,3e = 3.1 -
Hz, H-20), 3.86 (dd, 1H, H-3), 3.82 (dd, 1H, J5,6a = 7.7 Hz,
J5,6b = 4.1 Hz, H-5), 3.71 (dd, 1H, J6a,6b = 11.0 Hz, H-6a), 3.68 (dd,
0
0
0
0
0
0
1H, H-6b), 3.55 (ddd, 1H, J4 ,3a = 11.8 Hz, J4 ,5 = 10.0 Hz, J4 ,3e = 4.7 -
Hz, H-40), 3.56 (s, 3H, C-1-OCH3), 3.45–3.42 (m, 1H, H-50), 3.42 (s,
3H, C-6-OCH3), 2.16 (ddd, 1H, J3e ,3a = 14.0 Hz, H-3e0), 2.02 (s, 3H,
0
0
NHCOCH3), 1.65 (ddd, 1H, H-3a0), 1.27 (d, 3H, J6 ,5 = 6.2 Hz, H-60).
13C NMR (125 M Hz, CDCl3): d 175.6 (NHCOCH3), 103.4 (C-10),
102.9 (C-1), 80.0 (C-3), 76.8 (C-50), 74.0 (C-5), 72.5 (C-6), 69.3 (C-
4), 68.5 (C-20), 67.7 (C-40), 59.4 (C-6-OCH3), 57.9 (C-1-OCH3), 52.1
(C-2), 37.3 (C-30), 23.0 (NHCOCH3), 18.0 (C-60). ES HRMS calcd for
ane (3.7 lL, 0.060 mmol) were added to the solution, which was
0
0
heated at 50 °C for 18 h. DMF was evaporated under high vacuo,
and the mixture was purified by HPLC using a Beckman C18 silica
column with a MeOH–H2O gradient to afford final compound 7
as a white solid (3.5 mg, 42%) and starting material 5 was recov-
C
16H29NO9Na (M+Na): 402.1735, found: 402.1730.
ered (4.0 mg, 50%). The data for compound 7: ½a D22
ꢂ17.3 (c 1.6,
ꢃ
1
CH3OH). H NMR (600 MHz, D2O): d 4.75 (s, 1H, H-10), 4.44 (d,
1H, J1,2 = 8.7 Hz, H-1), 4.10 (d, 1H, J4,3 = 3.0 Hz, H-4), 4.01 (dd, 1H,
J2,3 = 10.8 Hz, H-2), 3.82 (dd, 1H, J5,6a = 7.4 Hz, J5,6b = 3.2 Hz, H-5),
3.80 (dd, 1H, H-3), 3.72 (dd, 1H, J6a,6b = 10.9 Hz, H-6a), 3.68 (dd,
3.32. Methyl 2-amino-3-O-(3,6-dideoxy-b-
D-arabino-
hexopyranosyl)-2-deoxy- 6-O-methyl-b- -galactopyranoside (3)
D
0
0
0
0
Compound 2 (5.7 mg, 0.016 mmol) was dissolved in an etha-
nolic solution of potassium hydroxide (1 mL, 4 M) and the solution
was heated to reflux for 24 h. The reaction was stopped by removal
of heat, and ethanol was evaporated. The crude solid was applied
to a C18 Sep-Pak cartridge, followed by HPLC on C18 silica column
eluted with methanol–water system to afford the amino sugar 3
as a white solid (4.8 mg, 100% after recovery of starting material):
1H, H-6b), 3.58 (dd, 1H, J2 ,3a = 2.6 Hz, J2 ,3e = 2.6 Hz, H-20), 3.51
(s, 3H, C-1-OCH3), 3.45–3.43 (m, 2H, H-40, H-50), 3.42 (s, 3H, C-6-
OCH3), 3.41 (s, 3H, C-20-OCH3), 2.36 (ddd, 1H, J3e ,3a = 14.1 Hz,
0
0
J3e ,4 = 3.4 Hz, H-3e0), 2.02 (s, 3H, NHCOCH3), 1.53 (ddd, 1H,
0
0
J3a ,4 = 2.7 Hz, H-3a0), 1.26 (d, 3H, J6 ,5 = 5.7 Hz, H-60). 13C NMR
(125 MHz, CDCl3): d 175.3 (NHCOCH3), 103.9 (C-10), 102.9 (C-1),
80.9 (C-3), 77.9 (C-20), 77.0 (C-50), 73.9 (C-5), 72.6 (C-6), 69.0
0
0
0
0

L. Cui et al. / Carbohydrate Research 383 (2014) 1–13
13
(C-4), 67.8 (C-40), 59.4 (C-1-OCH3), 58.3 (C-20-OCH3), 57.9 (C-6-
OCH3), 51.9 (C-2), 34.6 (C-30), 23.0 (NHCOCH3), 18.0 (C-60). ES
HRMS calcd for C17H31NO9Na (M+Na): 416.1891, found: 416.1891.
3. Nitz, M.; Bundle, D. R. J. Org. Chem. 2000, 65, 3064–3073.
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Acknowledgments
9. Ferrier, R. J.; Furneaux, R. H. Carbohydr. Res. 1976, 52, 63–68.
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12. Cai, Y.; Ling, C. C.; Bundle, D. R. Org. Lett. 2005, 7, 4021–4024.
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15. Deslongchamps, P. Stereoelectronic Effects in Organic Chemistry; Pergamon
Press: Oxford, 1983.
This work was supported by the research grants of Professor
D.R.B. from the Alberta Glycomics Centre, formerly Alberta
Ingenuity Center for Carbohydrate Science (AICCS). We thank Dr.
Albin Otter, Dr. Thomas Peters, and Dr. Scott McGavin for parame-
ter setup in the STD NMR experiments.
16. Bouvet, V. R.; Ben, R. N. J. Org. Chem. 2006, 71, 3619–3622.
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9294.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2013.10.
012.
22. Hanessian, S.; Plessas, N. R. J. Org. Chem. 1969, 34, 1045–1053.
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