Synthesis of a Forssman antigen derivative for use in a conjugate vaccine

Article abstract of DOI:10.1016/j.carres.2011.09.015

The total chemical synthesis of a Forssman antigen analog is described. The pentasaccharide contains a functionalized tether which should facilitate future conjugation with immunogenic proteins. We found that the total synthesis can be efficiently achieved by following a convergent 2+3 strategy, and using N-Troc protected GalNAc thioglycoside as a donor.

Full text of DOI:10.1016/j.carres.2011.09.015

Carbohydrate Research 346 (2011) 2650–2662
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of a Forssman antigen derivative for use in a conjugate vaccine
⇑
Jianhao Feng, Rachel Hevey, Chang-Chun Ling
Alberta Ingenuity Center for Carbohydrate Science, Department of Chemistry, University of Calgary, Calgary AB, Canada T2N 1N4
a r t i c l e i n f o
a b s t r a c t
Article history:
The total chemical synthesis of a Forssman antigen analog is described. The pentasaccharide contains a
functionalized tether which should facilitate future conjugation with immunogenic proteins. We found
that the total synthesis can be efficiently achieved by following a convergent 2+3 strategy, and using
N-Troc protected GalNAc thioglycoside as a donor.
Received 18 August 2011
Received in revised form 15 September
2011
Accepted 17 September 2011
Available online 29 September 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Forssman antigen
Oligosaccharides
Total synthesis
Glycosylation
1. Introduction
saccharide as a tumor-specific antigen makes it an interesting can-
didate for cancer therapy. An analogous target pentasaccharide
The differential expression of carbohydrates on cell surfaces is
widely recognized to be an integral component of many biologi-
cal pathways and recognition processes. One such example is the
which incorporated a 6-aminohexyl linker at the reducing end
(2) was designed as a synthetic target; such a linker would facili-
tate conjugation with an immunogenic protein or peptide that
would be capable of generating the desired T-cell dependant im-
mune response (Fig. 1).
glycolipid Forssman antigen,
aGalNAc(1?3)bGalNAc(1?3)
aGal(1?4)bGal(1?4)bGal(1?4)bGlc1-ceramide (1), an example
of a sphingolipid which is an important class of glycolipids univer-
sally expressed in higher organisms such as humans, but typically
not in lower organisms such as bacteria and yeast (Fig. 1). The
Forssman antigen is a pentasaccharide member of the globo-series
of glycosphingolipids, and very closely resembles the Globo H anti-
gen as they share an identical Gb4 tetrasaccharide core.
Although a few syntheses of the antigen were already available
in the literature,4–6 none of the established methods had integrated
a linker molecule onto the oligosaccharide. In addition, we sought
to improve some of the limitations of the other methods. A chem-
ical method was chosen over an enzymatic approach in order to be
less restricted by the high substrate specificity,7–9 increased cost of
glycosyl donors, and the limited number of glycosyltransferases
available from commercial sources, commonly associated with
enzymatic syntheses. A key consideration in the chemical synthe-
Initial studies dating back to 1911 demonstrated that expres-
sion of the Forssman antigen is species specific: the antigen is ex-
pressed in some mammals (e.g., sheep, dogs, horses, chickens, etc.)
but not in others (rabbits, pigs, humans, etc.), referred to as Forss-
man positive species and Forssman negative species, respectively.1
Forssman negative species instead express the tetrasaccharide pre-
cursor (Gb4), which is a substrate for the final enzyme in the bio-
synthetic pathway, a b(1?3)GalNAc transferase referred to as
Forssman synthetase; the human genome contains the gene for
this synthetase but it is not actively expressed.2 Together with
playing an important role on the surface of Forssman positive
mammalian cells as well as viral coats, interestingly the Forssman
antigen is also present in several forms of human cancers (e.g., gas-
tric, colon, lung).3 For individuals with Forssman positive tumors,
chemical and immunological detection has confirmed the absence
of this antigen on their healthy tissues.2 The identity of this penta-
sis involves the stereocontrol of the
a- and b-linkages at the Gal-
NAc residues. The glycan fragment of the Forssman antigen was
first published by Paulsen et al. in 1980, in which glycosyl
bromides and a 2+3 synthetic approach were utilized.4 This was
followed by Ogawa’s group in 1989 synthesizing the glycosphingo-
lipid in its entirety, utilizing an azide/silyl ether combination as a
ceramide synthon and a glycopentaosyl fluoride donor.5 An alter-
native chemical synthesis was published by Magnusson et al. in
1994, which utilized AgOTf-mediated glycosylations to afford the
pertinent a
-linkage at the non-reducing end.6
2. Results and discussion
In an attempt to develop a more efficient chemical synthesis,
we envisioned using an exceptionally stable glycosyl donor (as
compared to Paulsen’s and Magnusson’s glycosyl halides) and to
⇑
Corresponding author.
E-mail address: ccling@ucalgary.ca (C.-C. Ling).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.09.015

J. Feng et al. / Carbohydrate Research 346 (2011) 2650–2662
2651
HO
convergent 2+3 approach (Fig. 2). The first strategy relied on a 2,3-
oxazolidinone group on the sugar at the terminal non-reducing po-
sition (4) in order to direct the desired a-linkage, since it had been
OH
O
OH
O
HO
HO
OH
established in the literature to give the appropriate stereochemis-
try under particular activation conditions.10,11 An N-2,2,2-trichlo-
roethoxycarbonyl (Troc) protected 2-amino group was envisioned
to direct the b-linkage via anchimeric assistance (5), and a general
advantage of this strategy was that it used only monosaccharide
donors (more easily obtained and synthesized at a lower cost),
their improved availability over more complex donors allowing
them to be used in excess to improve glycosylation yields. Alterna-
tively, the second strategy utilized the same trisaccharide core (6)
with an N-Troc group to direct the b-linkage, but coupled the ter-
minal non-reducing disaccharide (10) together before combining
it with the acceptor to offer a convergent approach. In principle,
AcHN
OH
O
O
O
NHAc
HO
O
OH
OH
O
HO
OR
O
HO
O
1 R = ceramide
2 R = (CH2)6NH2
OH
OH
Figure 1. Structure of Forssman antigen (1) and the designed synthetic analog (2)
for use in conjugate vaccines.
the
a(1?3)GalNAc linkage found in 10 could be obtained using
the protected glycosyl donor 4; however, we finally decided to em-
ploy the more accessible 2-azido imidate donor 11, due to poten-
tial problems that might occur during glycosylation between 4
decrease the number of multistep functional group transforma-
tions that were present throughout Ogawa’s synthesis. Two differ-
ent strategies were explored, an iterative 2+1+1+1 approach and a
HO
OH
O
HO OH
O
HO
OH
OH
O
AcHN
O
O
NHAc
HO
O
OH
O
OH
HO
O
O
NH2
6
HO
O
OH
OH
2
Strategy A
Strategy B
AcO
Ph
Ph
AcO OBn
OAc
Ph
Ph
O O
O
O
O
O
O
O
O
AcO
O O
O
O
N
AcHN
O
O
O
O
O
Ac
O
O
TrocNH
TrocNH
BnO
BnO
O
O
OBn
O
OBn
OBn
O
OBn
BnO
O
O
BnO
O
O
N3
O
N3
6
6
BnO
BnO
O
OBn
OBn
OBn
OBn
3
9
Ph
O O
HO
Ph
Ph
AcO
AcO
OAc
O
O
OBn
AcO
O
O
O
BnO
O
O
O
O
OBn
O
OBn
O
+
+
+
AcHN
O
SPhCl
BnO
O
O
O
N
AcO
SPhCl
O
N3
6
BnO
SPhCl
Ph
NHTroc
Ac
O
NHTroc
10
OBn
OBn
4
5
6
Ph
AcO
OAc
O
O
O
O
O
HO
BnO
OBn
+
OBn
NH
O
BnO
O
O
AcO
+
O
O
N3
SPhCl
NHTroc
6
HO
SPhCl
O
OBn
CCl3
AcO
N3
O
OBn
7OBn
11
12
8
Figure 2. Retrosynthesis for target (2): iterative approach (strategy A) and convergent 2+3 approach (strategy B).

2652
J. Feng et al. / Carbohydrate Research 346 (2011) 2650–2662
and 12, as both parts contain a thioglycoside functionality, and
compound 12 appeared to be more reactive than 4—this would
not provide us with the desired chemoselectivity. Our analysis con-
cluded that retaining the N-Troc protecting group in disaccharide
donor (10) is advantageous because this would render the thiodi-
saccharide 10 more reactive thus increasing the efficiency of the fi-
nal glycosylation step. The only downside of using the 2-azido
imidate 11 as a donor is that the glycosylation step would produce
an anomeric mixture, which we hoped to separate by careful
chromatography.
acetate via a nucleophilic substitution to afford the target building
block (4 ).
a
To obtain the N-Troc protected thioglycoside donor 5, we
started with compound 18 (Fig. 4), conveniently prepared from
N-acetyl-D
-glucosamine using our recently published method.19
The acyl groups in GalNAc derivative 18 were deprotected via an
overnight reflux in aqueous NaOH solution; after neutralization
with HCl, the mixture was concentrated and purified by column
chromatography to give 19 in excellent yield (94%). Treatment of
19 with TrocCl in the presence of triethylamine provided the crude
N-Troc protected triol intermediate 20, which was directly con-
verted to the 4,6-benzylidene acetal 12 in 51% yield over two steps.
A final acetylation afforded the orthogonally protected building
block 5 in almost quantitative yield.
The synthesis of thiogalactosyl donor 7 was achieved in five
steps from the previously known p-chlorophenyl thioglycoside
21 (Fig. 5). To introduce a non-participating group (Bn) at O-2 po-
sition as well as an orthogonal protecting group (Ac) at O-3, the
tetraol 2112 was first refluxed in neat 2,2-dimethoxypropane with
a catalytic amount of 10-camphorsulfonic acid (CSA) to form the
3,4-acetal and a mixed acetal at the O-6 position, which was then
used directly for the subsequent O-benzylation step to afford pro-
tected intermediate 22. The isopropylidene acetals were then fully
The desired oxazolidinone-protected GalNAc donor 4 was syn-
thesized over five steps (Fig. 3) beginning from the known partially
protected D
-glucosamine thioglycoside 13.12 Removal of the phtha-
limido protection using 1,2-ethylenediamine-mediated conditions
afforded the desired aminoalcohol 14 in 89% yield. A subsequent
installation of the oxazolidinone moiety was realized using p-
nitrophenyl chloroformate as a reagent; the amide bond formation
followed by a ring closure afforded the oxazolidinone derivative 15
in 88% yield. The 2-amido position of 15 was subsequently pro-
tected via an acetylation using NaH and acetyl chloride (?16,
91% yield), based on a method recently published by Crich.13 Using
either Ac2O/pyridine/4-dimethylaminopyridine or acetyl chloride/
N,N-diisopropylethylamine, compound 16 consistently resulted in
disappointingly low yields.14,15 A reductive regioselective acetal
opening was then performed using Et3SiH and BF3ꢀEt2O to afford
the free alcohol 17. Interestingly, the conditions used for the acetal
opening concomitantly resulted in scrambling of the anomeric cen-
PivO
HO
OPiv
O
OH
O
a
SPhCl
SPhCl
ter; the a-anomer 17a was isolated in 54% yield while the related
AcO
HO
b-isomer was obtained in 8% yield after column chromatography.
This is in agreement with similar observations made previously
by several other groups while performing acid-catalyzed acetal
openings on sugars protected with a 2,3-oxazolidinone moiety,
which has been supported by an endo-cleavage mecha-
19NRH=RH
18NHAc
b
20 R=Troc
c
nism.9,14,16–18 The
a-thioglycoside 17a was then used for triflation
Ph
Ph
of the free 4-OH position, followed by an inversion using sodium
O
O
O
O
O
O
d
SPhCl
O
O
Ph
Ph
SPhCl
AcO
HO
O
O
O
a
O
SPhCl
SPhCl
NHTroc
NHTroc
HO
HO
5
12
NPhth
NH2
14
b
13
Figure 4. Synthesis of galactosaminyl donor 5. (a) 1 M NaOH, reflux, o/n; (b) TrocCl,
NEt3, MeOH; (c) PhCH(OMe)2,CSA, DMF; (d) Ac2O, Py.
O
Ph
O
Ph
OMe
O
O
HO
OH
O
O
SPhCl
SPhCl
O
O
O
a,b
O
c
O
SPhCl
NH
N
HO
O
SPhCl
Ac
O
OH
21
O
O
15
16
22 OBn
c
d
Ph
OBn
AcO
O
BnO
O
O
O
N
HO
e-f
OH
O
HO
O
SPhCl
O
d
O
SPhCl
SPhCl
N
RO
HO
SPhCl
Ac
A4cα
OBn
OBn
O
O
17
23
24 R = H
7 R = Ac
e
Figure 3. Synthesis of the galactosaminyl donor (4a) containing 2,3-oxazolidinone
protection. (a) Ethylenediamine, EtOH, reflux; (b) p-nitrophenyl chloroformate,
NaHCO3, H2O/CH3CN; (c) AcCl, NaH, DMF, ꢁ10 °C; (d) Et3SiH, BF3ꢀEt2O, DCM; (e)
Tf2O, Py/DCM; (f) NaOAc, DMF, 60 °C.
Figure 5. Synthesis of the galactosyl donor 7. (a) 2,2-Dimethoxypropane,CSA; (b)
BnBr, NaH, DMF; (c) CH3CO2H/H2O; (d) PhCH(OMe)2, CSA, CH3CN; (e) Ac2O, Py.

J. Feng et al. / Carbohydrate Research 346 (2011) 2650–2662
2653
hydrolyzed to give the desired triol 23 in 52% yield over three
steps, and the 4,6-benzylidene acetal subsequently introduced in
83% yield (?24). After a final acetylation step, the desired orthog-
onally protected building block (7) was obtained (95% yield).
Synthesis of the lactosyl acceptor 8 began via the glycosylation
of perbenzoylated lactosyl bromide (25) with the 6-azido-hexan-1-
ol using AgOTf promotion (Fig. 6);20 the desired b-glycoside (26)
was obtained in excellent yield (94%). Zemplén transesterification
afforded the fully debenzoylated intermediate (27, 97% yield),
which was then protected with a 4,6-benzylidene acetal (?28,
79% yield). Following a per-O-benzylation (29, 84% yield), a regio-
selective reductive opening of the acetal was achieved using Et3SiH
and BF3ꢀEt2O,21 to afford the desired acceptor (8). When the reac-
tion was carried out at room temperature, the majority of the start-
BzO
Br
BzO
OBz
O
OBz
O
OBz
BzO
O
BzO
O
BzO
O
a
O
N3
6
BzO
BzO
O
OBz 26
OBz 25
OBz
OBz
Ph
b
O
O
OH
O
HO
OH
O
OH
O
HO
HO
O
N3
O
N3
6
O
6
O
HO
BnO
c
O
OH
OH
OH
OH
28
d
27
Ph
O
HO
OBn
O
OBn
OBn
O
O
BnO
O
BnO
O
N3
O
N3
6
O
6
e
BnO
BnO
O
O
OBn
OBn29
8
OBn
OBn
Figure 6. Synthesis of the lactosyl acceptor 8. (a) 6-Azidohexan-1-ol, AgOTf, toluene; (b) NaOMe, MeOH; (c) PhCH(OMe)2, CSA, DMF; (d) BnCl, NaH, DMF; (e) BF3ꢀEt2O, Et3SiH,
CH2Cl2.
Ph
Ph
Ph
O O
O
O O
O
RO
O
O
O
5
AcO
7
O
a
NHTroc
c
BnO
+
8
O
OBn
BnO
OBn
O
O
OBn
O
BnO
O
OBn
O
O
N3
6
BnO
O
BnO
O
O
N3
6
BnO
OBn
OBn
OBn
31 R = Ac
30 R=Ac
6 R=H
OBn
b
b
OBn
Ph
Ph
AcO
O
Ph
O O
Ph
O
O
O
O
O
4α
O O
O
O
N
c
O
O
O
HO
O
Ac
O
O
NH
O
NH
O
O
BnO
O
BnO
O
OMe
OMe
OBn
OBn
O
OBn
O
OBn
O
O
BnO
O
BnO
O
N3
O
N3
6
6
BnO
BnO
OBn
OBn
OBn
OBn
33
32
Figure 7. Attempt at assembling the pentasaccharide using the 2+1+1+1 iterative approach. (a) TfOH, NIS, CH2Cl2; (b) NaOMe, MeOH/CH2Cl2; (c) TfOH, NIS, CH2Cl2.

2654
J. Feng et al. / Carbohydrate Research 346 (2011) 2650–2662
ing material was consistently found to be converted to a more po-
lar by-product, presumably as a result of reduction of the azido
group; when we kept the reaction at 0 °C overnight, the desired
compound 8 was obtained as a majority and isolated in 56% yield
by column chromatography on silica gel.
tunately multiple attempts at this final glycosylation step using
NIS/TfOH activation were unsuccessful in delivering the desired
product, as only decomposition products were obtained. As a re-
sult, our attention turned to the alternative convergent 2+3
approach.
An initial attempt of assembling the building blocks in the iter-
ative approach began by coupling lactosyl acceptor 8 to thiogalac-
tosyl donor 7; using N-iodosuccinimide/triflic acid (NIS/TfOH)
promotion the desired trisaccharide 30 was obtained in high yield
The key to succeed in the second approach was to prepare the
(1?3)-linked di-galactosamine donor 10. The imidate building
a
block 11 was first synthesized from thioglycoside intermediate
19 that had been previously generated during the synthesis of
building block 5 (see Fig. 4). The free 2-amino group was converted
to an azide using Tf2O and NaN3 in pyridine (34), based on a meth-
od introduced by C.-H. Wong’s group23 and later improved by Ye
et al. (Fig. 8).24 The crude material was then acetylated (35) and
the anomeric thiophenyl group hydrolyzed using NIS and CH3CN/
H2O to afford the desired glycosyl hemiacetal (36), which was di-
rectly converted into the imidate (11)25 by reacting with trichloro-
acetonitrile in the presence of K2CO3 in anhydrous CH2Cl2.
Coupling between this donor and acceptor 12 proceeded smoothly
upon activation with trimethylsilyl triflate to afford the product as
(87%) and excellent anomeric selectivity, as the
a-isomer 30 was
the only product isolated from the reaction (Fig. 7). Deacetylation
to reveal the free OH afforded the required acceptor 6 in almost
quantitative yield (97%). The coupling between the obtained alco-
hol 6 with building block 5 (2 equiv) proceeded with very high effi-
ciency to produce the desired tetrasaccharide 31 in 96% yield.
Unfortunately, attempts at selectively removing the OAc protecting
group at the non-reducing end without affecting the Troc group
proved to be more difficult than expected. For example, when
using guanidine/guanidinium buffer in anhydrous methanol, the
reaction was found to be sluggish;22 however, when using more
forcing conditions (catalytic NaOMe in MeOH/CH2Cl2) the reaction
resulted in a complex product mixture. For the latter case, the
major product was isolated in 47% yield and characterized to be
compound 32, which had the expected O-deacetylation at the
non-reducing end galactosamine residue but unfortunately the
N-Troc group had also been converted to the corresponding methyl
carbamate via transesterification. Since the methyl carbamate
could later be deprotected using saponification, we attempted to
an anomeric mixture, of which the desired a-anomer (37) was ob-
tained in 57% yield by column chromatography. A recent paper has
suggested that the OAc protecting groups at C-3 and C-4 improve
the
studies suggest that transition state stabilities direct formation of
the
-anomer.26 Since the azide moiety needed to be differentiated
a-selectivity of the glycosylation reaction, as computational
a
from the azide at the linker terminus in the final product, we there-
fore converted it directly to the corresponding NHAc group prior to
the final glycosylation step. The transformation was realized using
neat thioacetic acid as a reagent based on a literature procedure,27
and the desired disaccharide donor 10 was obtained in almost
quantitative yield (98%).
synthesize the pentasaccharide 33 by reacting 32 with 4
a
. Unfor-
With the desired disaccharide donor 10 and trisaccharide
acceptor 6 in hand, the final glycosylation reaction was performed
via NIS/TfOH activation (Fig. 9) using the acceptor 6 in slight excess
(1.2 equiv). The glycosylation started at ꢁ78 °C and the tempera-
ture was raised to ꢁ30 °C; after 1 h, the desired pentasaccharide
was isolated in excellent yield (89%). This demonstrates the high
glycosylation efficiency of disaccharide donor 10.
The identity of the final protected pentasaccharide (9) was con-
firmed by high-resolution mass spectrometry and one/two-dimen-
sional NMR studies. For example, high resolution electrospray
mass spectrometry showed a peak with m/z 2122.7337, which cor-
responds to the [M+Na]+ adduct. In the HSQC spectra (Fig. 10), the
HO
AcO
OH
OAc
O
O
a
b
SPhCl
SPhCl
HO
AcO
19
N3
N3
34
35
c,d
Ph
AcO
OAc
O
12
AcO
OAc
O
O
O
AcO
e
O
AcO
R
SPhCl
O
N3 OR
NHTroc
C–H correlations indicated that two anomeric
a-linkages were
36 R = H
11 R = C(=NH)CCl3
37 R = N3
10 R = NHAc
present (1H: 4.9–5.2 ppm 13C: 93–102 ppm) and three anomeric
b-linkages were present (1H: 4.3–4.6 ppm, 13C: 100–105 ppm);
the two benzylidene acetal C–H peaks were also observed. Two
characteristic N-attached C–H bonds were present (4.55 pm/
46 ppm and 4.10 ppm/53 ppm, 1H/13C, respectively), as was the
f
Figure 8. Synthesis of
a(1?3)-linked thiodisaccharide donor 10. (a) TfN3, CuSO4,
NEt3, Py; (b) Ac2O, Py; (c) NIS, CH3CN/H2O; (d) K2CO3, CCl3CN/CH2Cl2; (e) TMSOTf/
CH2Cl2; (f) AcSH.
Ph
AcO
OAc
O
Ph
O
O
AcO
O O
O
AcHN
O
O
O
NHTroc
a
6 + 10
BnO
O
OBn
OBn
O
O
BnO
O
O
N3
6
BnO
OBn
OBn
9
Figure 9. Synthesis of the fully protected pentasaccharide 9 via 2+3 assembly. (a) TfOH, NIS, CH2Cl2.

J. Feng et al. / Carbohydrate Research 346 (2011) 2650–2662
2655
Figure 10. The 1H–13C HSQC spectra of fully protected pentasaccharide 9 in CDCl3 (400 MHz).
distinct signal from the methylene unit attached to the azide of the
linker (3.21 ppm/51 ppm, 1H/13C, respectively).
N-acetylated intermediate. However, the final hydrogenation dis-
appointingly afforded a complex mixture. The multistep depro-
tections appeared to be complicated by the benzylidene groups
which were removed by the hydrogenation at a much slower
rate than the benzyl ethers. In an alternative approach
(Fig. 11), an acid-catalyzed hydrolysis was first performed to re-
move the 4,6-benzylidene acetals, followed by a Zemplén transe-
Initial attempts to deprotect the fully-protected pentasaccha-
ride began with Zemplén transesterification, which was expected
to remove all the O-acetyl groups and concomitantly convert the
Troc group to the methyl carbamate; after another base-
catalyzed saponification step to remove the carbamate, the mix-
ture was neutralized with acetic anhydride to afford the desired
sterification step of the crude material and
a subsequent
HO
OH
O
HO
OH
O
HO
OH
AcHN
OH
O
O
O
a-e
AcHN
9
BnO
O
OBn
O
OBn
O
BnO
O
O
N3
6
BnO
38
OBn
OBn
f
HO
OH
O
HO
OH
O
HO
OH
AcHN
OH
O
O
O
AcHN
HO
O
OH
O
OH
O
HO
O
O
NH2.AcOH
6
HO
OH
OH
2
Figure 11. Deprotection of pentasaccharide 9 to obtain the desired Forssman antigen analog (2). (a) 80% AcOH/H2O, 90 °C; (b) NaOMe, MeOH, 50 °C; (c) 2 N NaOH, reflux; (d)
Ac2O, Py; (e) NaOMe, MeOH; (f) H2, 20% Pd(OH)2-C, MeOH/H2O/AcOH.

2656
J. Feng et al. / Carbohydrate Research 346 (2011) 2650–2662
saponification of the carbamate as before; the crude mixture was
then concentrated, fully acetylated, and then de-O-acetylated
using NaOMe in MeOH to afford mainly the desired product
(38). The final hydrogenation reaction was performed to afford
2 mg of the desired product 2 (11.3% yield from the fully pro-
tected pentasaccharide).
4.2. p-Chlorophenyl 2-amino-4,6-O-benzylidene-2-deoxy-1-
thio-b- -glucopyranoside (14)
D
Compound 1312 (8.1 g, 15.5 mmol) was dissolved in absolute
ethanol (80 mL) and ethylene diamine (20.0 mL, 310 mmol) was
added to the suspension. The reaction was refluxed at 95 °C over-
night. HCl (2 N) was then used to neutralize the reaction to modify
the pH to ꢃ8 and the mixture was concentrated. The mixture was
diluted by EtOAc, and washed by water and brine. The organic
layer was dried over anhydrous Na2SO4 and evaporated to give
the compound 14 (5.44 g, 89.3% yield). Rf: 0.13 (EtOAc/toluene
The 1H–13C HSQC NMR spectrum of the final compound (2)
clearly confirmed the presence of five sets of anomeric C–H cor-
relation peaks at (5.10 ppm, 93.4 ppm), (4.96 ppm, 102.6 ppm),
(4.74 ppm, 100.3 ppm), (4.54 ppm, 103.2 ppm), and (4.51 ppm,
101.9 ppm), confirming that the obtained compound is a penta-
saccharide. The anomeric protons at 5.10 and 4.96 ppm appear
as doublets and have small coupling constants (<4 Hz), indicating
4:6). [a]
+23.7 (c 1.5, CHCl3). 1H NMR (400 MHz, CDCl3) dH 7.56–
D
7.28 (m, 9H, Ph), 5.55 (s, 1H, PhCH), 4.52 (d, 1H, J = 9.8 Hz, H-1),
4.36 (dd, 1H, J = 10.5, 4.4, H-6a), 3.79 (dd, 1H, J = 9.9, 1.3 Hz, H-
6b), 3.64 (dd, 1H, J = 8.6, 8.6 Hz, H-3), 3.57–3.46 (m, 2H, H-4 + H-
5), 2.79 (dd, 1H, J = 9.4, 9.4 Hz, H-2), 2.02 (s, 3H, OH + NH2). 13C
NMR (100 MHz, CDCl3) dC 136.97 (Ph), 134.18 (Ph), 130.30 (Ph),
129.33 (Ph), 129.24 (Ph), 128.37 (Ph), 126.27 (Ph), 101.92 (PhCH),
90.00 (C-1), 80.88 (C-5), 74.79 (C-3), 70.53 (C-4), 68.58 (C-6),
that they both have an
suggests that they correspond to the anomeric protons of the
GalNAc and Gal, respectively. The three remaining anomeric
a-anomeric configuration; HSQC spectra
a
a
protons also appear as doublets but with a large coupling con-
stant (>7 Hz), confirming that they all have a b-anomeric config-
uration, with the anomeric proton resonating at 4.74 ppm
corresponding to the bGalNAc residue, while those protons at
4.54 ppm, 4.51 ppm correspond to the bGal and bGlc residues,
respectively. The structure of 2 was finally confirmed by high res-
olution electrospray mass spectrometry which showed a peak
with m/z 1010.4390, which correlates with the expected [M+H]+
adduct.
56.47 (C-2). HRESIMS: Calcd for
416.0694. Found: 416.0679.
C
19H20NO4Na (M+Na+): m/z
4.3. p-Chlorophenyl 4,6-O-benzylidene-2-deoxy-2,3-N,O-
carbonyl-1-thio-b- -glucopyranoside (15)
D
Compound 14 (4.28 g, 10.9 mmol) was added into a mixture of
saturated NaHCO3 (60 mL) and CH3CN (60 mL) and stirred at 0 °C.
p-Nitrophenyl chloroformate (4.6 g in 10 mL CH3CN, 21.7 mmol)
was added into the mixture. Then reaction was kept at 0 °C for
2 h and warmed to rt overnight. The mixture was then diluted by
EtOAc and washed with H2O. The organic layer was dried over
anhydrous Na2SO4 and evaporated. The residue was purified by
column chromatography on silica gel using 4% EtOAc–toluene as
eluent to furnish the compound 15 (4.0 g, 87.7% yield). Rf: 0.35
3. Conclusions
Since the Forssman antigen is a tumor-specific antigen closely
integrated with cancer biology, it is desirable to have it readily
accessible in order to incorporate it into carbohydrate conjugate-
vaccines used to target different types of cancer. It could become
immensely valuable if able to effectively raise an immune response
against the Forssman pentasaccharide expressed on the surface of
cancer cells. Although improvements need to be made to the
deprotection sequence, we have successfully developed a synthetic
methodology that allows for the incorporation of a linker at the
reducing end of the pentasaccharide, in order to eventually conju-
gate it to an immunogenic protein or peptide fragment required for
an effective vaccine.
(EtOAc/toluene 1:4). [a]
+15.1 (c 1.8, CHCl3). 1H NMR (400 MHz,
D
CDCl3) dH 7.51–7.45 (m, 4H, SPhCl), 7.42–7.33 (m, 5H, benzyli-
dene_Ph), 5.60 (s, 1H, PhCH), 5.46–5.37 (m, 1H, NH), 4.82 (d, 1H,
J = 9.8, H-1), 4.43–4.34 (m, 2H, H-6a + H-3), 4.06–3.98 (dd, 1H,
J = 9.5, 8.7 Hz, H-4), 3.92 (dd, 1H, J = 10.4, 10.4 Hz, H-6b), 3.64
(ddd, 1H, J = 10.1, 8.6, 4.7, H-5), 3.46 (m, 1H, H-2). 13C NMR
(100 MHz, CDCl3) dC 158.15 (oxazolidinone), 136.25 (Ph), 135.72
(Ph), 134.99 (Ph), 129.59 (Ph), 129.35 (Ph), 128.32 (Ph), 128.20
(Ph), 126.08 (Ph), 101.44 (PhCH), 85.24 (C-1), 80.53 (C-3), 78.58
(C-4), 73.26 (C-5), 68.24 (C-6), 59.55 (C-2). HREIMS: Calcd for
4. Experimental data
4.1. General methods
C
20H18NO5SCl (M+): m/z 419.0594. Found: 419.0595.
Optical rotations were determined in a 5-cm cell at 25 2 °C.
4.4. p-Chlorophenyl 2-acetamido-4,6-O-benzylidene-2-deoxy-
2,3-N,O-carbonyl-1-thio-b- -glucopyranoside (16)
½
a 2D5
ꢂ
values are given in units of 10ꢁ1 deg cm2 gꢁ1. Analytical TLC
D
was performed on Silica Gel 60-F254 (E. Merck, Darmstadt) with
detection by quenching of fluorescence and/or by charring with
5% sulfuric acid in water or with a ceric ammonium molybdate
dip. All commercial reagents were used as supplied unless other-
wise stated. Column chromatography was performed on Silica
Gel 60 (Silicycle, Ontario). Organic solutions from extractions were
dried with anhydrous Na2SO4 prior to concentration under vacuum
at <60 °C (bath). NMR spectra were recorded at 400 MHz on Bruker
spectrometers. The first-order proton chemical shifts dH and dC are
reported in d (ppm) for solutions in CDCl3 and referenced to resid-
ual CHCl3 (dH 7.27 and dC 77.0). 1H and 13C NMR spectra were as-
signed with the assistance of 2D GCOSY, 2D TOCSY, 2D GHSQC,
2D GHMBC, or 1D TOSCY experiments. High-resolution ESI-QTOF
mass spectra were recorded on an Agilent 6520 Accurate Mass
Quadrupole Time-of-Flight LC/MS spectrometer. Elemental analy-
ses were obtained with a Perkin Elmer Series II 2400 CHNS/O Ana-
lyzer. All of the data were obtained by the analytical services of the
Department of Chemistry, University of Calgary.
Compound 15 (3.0 g, 7.14 mmol) was dissolved in anhydrous
DMF (20 mL) with 60% NaH (1.0 g, 41.7 mmol). Acetyl chloride
(2.0 mL, 28.6 mmol) was then slowly added into the mixture at
ꢁ10 °C and the reaction was stirred at rt overnight. NaHCO3 was
added and the mixture was extracted by CH2Cl2 and H2O. The or-
ganic layer was dried over anhydrous Na2SO4 and evaporated.
The residue was purified by column chromatography on silica gel
using 1% EtOAc–CH2Cl2 as eluent to give 16 (3.0 g, 90.9% yield).
Rf: 0.60 (EtOAc/toluene 1:4). [
a]
+36.9 (c 1.4, CHCl3). 1H NMR
D
(400 MHz, DMSO-d6) dH 7.46–7.35 (m, 9H, Ph), 5.76 (s, 1H, PhCH),
5.55 (d, 1H, J = 8.8 Hz, H-1), 4.80 (dd, 1H, J = 10.5, 10.5 Hz, H-3),
4.31–4.22 (m, 2H, H-4 + H-2), 4.19 (dd, 1H, J = 10.0, 4.6 Hz, H-6a),
3.91 (dd, 1H, J = 10.2, 10.2 Hz, H-6b), 3.78 (ddd, 1H, J = 10.1, 8.4,
4.6 Hz, H-5), 2.44 (s, 3H, Ac). 13C NMR (100 MHz, DMSO-d6) dC
173.01 (Ac), 154.20 (oxazolidinone), 137.42 (Ph), 134.37 (Ph),
131.93 (Ph), 131.32 (Ph), 129.61 (Ph), 129.42 (Ph), 128.67 (Ph),
126.67 (Ph), 100.86 (PhCH), 85.41 (C-1), 78.26 (C-4), 77.38 (C-3),

J. Feng et al. / Carbohydrate Research 346 (2011) 2650–2662
2657
72.54 (C-5), 67.72 (C-6), 60.44 (C-2), 24.86 (Ac). HREIMS: Calcd for
22H20NO6SCl (M+): m/z 461.0700. Found: 461.0719.
(400 MHz, CDCl3) dH 7.45–7.40 (m, 2H, SPhCl), 7.35 (m, 5H, Bn),
7.24–7.19 (m, 2H, SPhCl), 6.17 (d, 1H, J = 3.8 Hz, H-1), 5.72 (dd,
1H, J = 1.2, <1 Hz, H-4), 4.61–4.41 (m, 5H, H-3 + H-2 + H-
5 + Bn_CH2), 3.68–3.49 (m, 2H, H-6a + H-6b), 2.54 (s, 3H, Ac),
2.08 (s, 3H, Ac). 13C NMR (100 MHz, CDCl3) dC 171.43 (Ac),
169.18 (Ac), 152.45 (oxazolidinone), 137.43 (Ph), 134.76 (Ph),
134.36 (Ph), 130.35 (Ph), 129.36 (Ph), 128.48 (Ph), 127.95 (Ph),
127.84 (Ph), 87.07 (C-1), 74.32 (C-5), 73.65 (Bn_CH2), 70.12 (C-3),
67.99 (C-6), 65.85 (C-4), 56.03 (C-2), 23.87 (Ac), 20.55 (Ac). HRC-
IMS: Calcd for C24H28N2O7SCl (M+NH4þ): m/z 523.1306. Found:
523.1330. Anal. Calcd for C24H24NO7SCl: C, 56.97; H, 4.78; N,
2.77. Found: C, 57.41; H, 4.78; N, 2.70.
C
4.5. p-Chlorophenyl 2-acetamido-6-O-benzyl-2-deoxy-2,3-N,O-
carbonyl-1-thio- ,b- -glucopyranoside (17)
a
D
Triethylsilane (3.65 mL, 22.8 mmol) and BF3ꢀEt2O (359
lL,
2.85 mmol) were added to a suspension of 16 (880 mg, 1.9 mmol)
in anhydrous CH2Cl2 (30 mL) at 0 °C under argon. The reaction was
stirred at 0 °C for 2 h and then diluted by CH2Cl2 and washed with
H2O. The organic layer was dried over anhydrous Na2SO4 and evap-
orated. The residue was purified by column chromatography on sil-
ica gel using 9% EtOAc–toluene as eluent to produce 17
a (481 mg,
54.4% yield) and 17b (67 mg, 8.3% yield).
4.7. p-Chlorophenyl 2-amino-2-deoxy-1-thio-b-D-
galactopyranoside (19)
4.5.1. p-Chlorophenyl 2-acetamido-6-O-benzyl-2-deoxy-2,3-
N,O-carbonyl-1-thio- -glucopyranoside (17
Rf: 0.35 (EtOAc/toluene 1:4). [
D +204.1 (c 1.1, CHCl3). 1H NMR
a-
D
a)
Compound 18 (10.4 g, 18.6 mmol) was stirred in aq NaOH
(500 mL, 1 N) and the suspension was refluxed overnight. HCl
(2 N) was added to modify the pH to 8. Then the solvent was evap-
orated and MeOH was added to wash the residue. The precipitate
was filtered and purified by flash column chromatography on silica
gel using CH2Cl2/MeOH/H2O 80:20:2 as eluent to give the com-
a
]
(400 MHz, CDCl3) dH 7.43–7.39 (m, 2H, SPhCl), 7.38–7.29 (m, 5H,
Bn), 7.28–7.22 (m, 2H, SPhCl), 6.12 (d, 1H, J = 4.4 Hz, H-1), 4.63
(d, 1H, J = 11.9 Hz, Bn_CH2), 4.55 (d, 1H, J = 11.9 Hz, Bn_CH2), 4.36
(dd, 1H, J = 12.1, 9.5 Hz, H-3), 4.16 (ddd, 1H, J = 8.4, 4.1, 4.1 Hz,
H-5), 4.10 (dd, 1H, J = 9.3, 3.3 Hz, H-4), 4.04 (dd, 1H, J = 12.2,
4.5 Hz, H-2), 3.86 (dd, 1H, J = 10.5, 4.4 Hz, H-6a), 3.77 (dd, 1H,
J = 10.5, 3.9 Hz, H-6b), 3.46 (d, 1H, J = 3.3 Hz, OH-4), 2.53 (s, 3H,
Ac). 13C NMR (100 MHz, CDCl3) dC 171.28 (Ac), 153.02 (oxazolidi-
none), 137.38 (Ph), 134.57 (Ph), 133.96 (Ph), 130.92 (Ph), 129.36
(Ph), 128.57 (Ph), 128.07 (Ph), 127.78 (Ph), 86.34 (C-1), 78.30 (C-
3), 73.80 (Bn_CH2), 72.89 (C-5), 69.83 (C-4), 69.13 (C-6), 59.57
(C-2), 23.79 (Ac). HRESIMS: Calcd for C22H22NO6SClNa (M+Na+):
m/z 486.0748. Found: 486.0756. Anal. Calcd for C22H22NO6SCl: C,
56.96; H, 4.78; N, 3.02. Found: C, 57.10; H, 4.94; N, 3.00.
pound 19 (5.35 g, 94.1% yield). Rf: 0.30 (MeOH/CH2Cl2 2:8). [a]
D
ꢁ43.6 (c 5.2, MeOH). 1H NMR (400 MHz, CD3OD) dH 7.60–7.55
(m, 2H, Ph), 7.34–7.29 (m, 2H, Ph), 4.58 (d, 1H, J = 10.0 Hz, H-1),
3.85 (dd, 1H, J = 3.1, <1 Hz, H-4), 3.79 (dd, 1H, J = 11.5, 6.9 Hz, H-
6a), 3.72 (dd, 1H, J = 11.5, 5.1 Hz, H-6b), 3.59 (ddd, 1H, J = 6.7,
5.1, 1.0 Hz, H-5), 3.47 (dd, 1H, J = 9.8, 3.2 Hz, H-3), 3.04 (dd, 1H,
J = 9.9, 9.9 Hz, H-2). 13C NMR (100 MHz, CD3OD) dC 133.25 (Ph),
132.72 (Ph), 132.23 (Ph), 128.65 (Ph), 88.25 (C-1), 79.45 (C-5),
73.98 (C-3), 68.11 (C-4), 61.36 (C-6), 51.75 (C-2). HRESIMS: Calcd
for C12H17NO4SCl (M+H+): m/z 306.0561. Found: 306.0556.
4.5.2. p-Chlorophenyl 2-acetamido-6-O-benzyl-2-deoxy-2,3-
4.8. p-Chlorophenyl 2-deoxy-2-N-(2,2,2-trichloroethoxycarbon-
N,O-carbonyl-1-thio-b-
D
-glucopyranoside (17b)
ylamino)-1-thio-b-D-galactopyranoside (20)
Rf: 0.27 (EtOAc/toluene 1:4). [
a]
ꢁ62.5 (c 2.1, CHCl3). 1H NMR
D
(400 MHz, CDCl3) dH 7.45–7.41 (m, 2H, SPhCl), 7.40–7.29 (m, 5H,
Bn), 7.22–7.17 (m, 2H, SPhCl), 4.83 (d, 1H, J = 8.4 Hz, H-1), 4.60–
4.52 (m, 2H, Bn_CH2), 4.19–3.99 (m, 3H, H-3 + H-4 + H-2), 3.82–
3.73 (m, 2H, H-6a + H-6b), 3.55 (ddd, 1H, J = 8.1, 5.0, 5.0 Hz, H-5),
3.20 (d, 1H, J = 2.0 Hz, OH-4), 2.57 (s, 3H, Ac). 13C NMR (100 MHz,
CDCl3) dC 173.10 (Ac), 153.75 (oxazolidinone), 137.25 (Ph),
134.27 (Ph), 133.83 (Ph), 132.24 (Ph), 129.00 (Ph), 128.60 (Ph),
128.12 (Ph), 127.85 (Ph), 87.00 (C-1), 81.64 (C-3), 79.53 (C-5),
73.85 (Bn_CH2), 69.79 (C-6), 69.77 (C-4), 59.57 (C-2), 24.69 (Ac).
HREIMS: Calcd for C22H22NO6SCl (M+): m/z 463.0856. Found:
463.0853. Anal. Calcd for C22H22NO6SCl: C, 56.96; H, 4.78; N,
3.02. Found: C, 57.00; H, 4.89; N, 2.95.
Compound 19 (700 mg, 2.29 mmol) was dissolved in a mixture
of anhydrous MeOH (6 mL) and Et3N (274.4 L, 2.98 mmol). 2,2,2-
Trichloroethoxycarbonyl chloride (461.8 L, 3.44 mmol) was
l
l
added and stirred at 0 °C and the reaction was stirred at rt over-
night. The solution was concentrated and H2O was then added
and precipitate formed. The solid was collected by the vacuum fil-
tration and a following evaporation with toluene gave the com-
pound 20. The crude product was used for next step without
further purification. Rf = 0.63 (MeOH/CH2Cl2 1.5:8.5).
4.9. p-Chlorophenyl 4,6-O-benzylidene-2-deoxy-2-N-(2,2,2-
trichloroethoxycarbonylamino)-1-thio-b-D-galactopyranoside
(12)
4.6. p-Chlorophenyl 2-acetamido-4-O-acetyl-6-O-benzyl-2-
deoxy-2,3-N,O-carbonyl-1-thio-
a
-D
-glucopyranoside (4
a)
PhCH(OMe)2 (436.6 lL, 2.91 mmol) and 10-camporsulfonic acid
were added to the solution of 20 (850 mg, crude) in anhydrous
DMF (3 mL). The reaction was stirred at rt overnight and Et3N
was then added to the mixture. The solvent was evaporated and
the residue was purified by column chromatography on silica gel
using 35% EtOAc–toluene as eluent to afford the compound 12
(662.1 mg, 50.8% yield over two steps). Rf: 0.67 (MeOH/CH2Cl2
Compound 17
ture of anhydrous CH2Cl2 (0.6 mL) and anhydrous pyridine
(0.6 mL). trifluoromethanesulfonic anhydride
At ꢁ10 °C,
(132.7 L, 0.78 mmol) was added and the reaction was stirred at
0 °C for 3.5 h. The solution was diluted by CH2Cl2 and washed with
H2O. The organic layer was dried over anhydrous Na2SO4 and evap-
orated below 40 °C to afford the crude triflate. This residue was
then dissolved in anhydrous DMF (1 mL) and NaOAc (215.7 mg,
2.6 mmol) was added. The reaction was stirred at 60 °C overnight.
The mixture was diluted by CH2Cl2 and washed by H2O. The organ-
ic layer was dried over anhydrous Na2SO4 and evaporated. The res-
idue was purified by column chromatography on silica gel using
a (122 mg, 0.26 mmol) was dissolved in a mix-
l
5:95). [a]
ꢁ48.5 (c 0.84, acetone). 1H NMR (400 MHz, acetone-
D
d6) dH 7.70–7.57 (m, 2H, SPh), 7.56–7.44 (m, 2H, benzylidene),
7.44–7.30 (m, 3H, benzylidene), 7.30–7.19 (m, 2H, SPh), 6.94 (d,
1H, J = 8.8 Hz, NH), 5.66 (s, 1H, PhCH), 4.94 (d, 1H, J = 9.7 Hz, H-
1), 4.86 (d, 1H, J = 12.3 Hz, Troc_CH2), 4.72 (d, 1H, J = 12.3 Hz,
Troc_CH2), 4.34 (dd, 1H, J = 2.3, <1 Hz, H-4), 4.22 (dd, 1H, J = 12.3,
1.6 Hz, H-6a), 4.17–4.11 (m, 2H, OH-3 + H-6b), 3.99–3.82 (m, 2H,
H-3 + H-2), 3.79–3.69 (m, 1H, H-5). 13C NMR (100 MHz, acetone-
d6) dC 154.51 (CO), 138.79 (Ph), 133.81 (Ph), 132.74 (Ph), 132.33
10% acetone–hexane as eluent to give 4
a (94 mg, 70.7% yield). Rf:
0.50 (EtOAc/toluene 1:4). [
a
]
D
+168.5 (c 2.1, CHCl3). 1H NMR

2658
J. Feng et al. / Carbohydrate Research 346 (2011) 2650–2662
(Ph), 128.63 (Ph), 128.59 (Ph), 127.83 (Ph), 126.53 (Ph), 100.56
(PhCH), 96.15 (CCl3), 85.78 (C-1), 75.65 (C-4), 73.93 (CH2_Troc),
71.42 (C-3), 70.06 (C-5), 69.01 (C-6), 53.16 (C-2). HREIMS: Calcd
for C16H17NO6Cl3 (MꢁSC6H4Cl): m/z 426.0092. Found: 426.0072.
and 10-camphorsulfonic acid were added. The reaction was stirred
for 30 min and Et3N was then added. The solvent was evaporated
and the residue was purified by column chromatography on silica
gel using 3% MeOH–CH2Cl2 as eluent to yield the compound 24
(3.50 g, 82.0% yield). Rf = 0.23 (EtOAc/Hexane 3:7). [
a
]
D
ꢁ6.22 (c
0.91, CHCl3). 1H NMR (400 MHz, CDCl3) dH 7.67–7.63 (m, 2H,
SPh), 7.52–7.28 (m, 10H, benzylidene + Bn), 7.23–7.18 (m, 2H,
SPh), 5.57 (s, 1H, PhCH), 4.76 (d, 1H, J = 10.7 Hz, PhCH2), 4.72 (d,
1H, J = 10.7 Hz, PhCH2), 4.59 (d, 1H, J = 9.4 Hz, H-1), 4.38 (dd, 1H,
J = 12.3, 1.4 Hz, H-6a), 4.22 (dd, 1H, J = 3.5, <1 Hz, H-4), 4.04 (dd,
1H, J = 12.4, 1.4 Hz, H-6b), 3.81 (dd, 1H, J = 9.0, 3.6 Hz, H-3), 3.61
(dd, 1H, J = 9.2, 9.2 Hz, H-2), 3.53–3.50 (m, 1H, H-5). 13C NMR
(100 MHz, CDCl3) dC 138.13 (Ph), 137.51 (Ph), 134.29 (Ph), 133.91
(Ph), 130.96 (Ph), 129.39 (Ph), 129.01 (Ph), 128.41 (Ph), 128.29
(Ph), 128.04 (Ph), 127.86 (Ph), 126.41 (Ph), 101.26 (PhCH), 85.83
(C-1), 77.00 (C-2), 75.64 (C-4), 75.28 (PhCH2), 74.37 (C-3), 69.82
(C-5), 69.19 (C-6). HREIMS: Calcd for C20H21O5 (MꢁSC6H5Cl+):
m/z 341.1389. Found: 341.1393.
4.10. p-Chlorophenyl 3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-
N-(2,2,2-trichloroethoxycarbonylamino)-1-thio-b-D-
galactopyranoside (5)
Compound 12 (50 mg, 0.088 mmol) was dissolved in a mixture
of pyridine (1.3 mL) and Ac2O (1.0 mL). The reaction was stirred at
rt overnight. The mixture was evaporated to dry to give the com-
pound 5 as a white solid (53 mg, 99% yield). Rf: 0.84 (MeOH/CH2Cl2
3:97). [
a]
ꢁ34.5 (c 0.91, CHCl3). 1H NMR (400 MHz, CDCl3) dH
D
7.58–7.67 (m, 2H, SPhCl), 7.35–7.45 (m, 5H, benzylidene), 7.15–
7.24 (m, 2H, SPhCl), 5.52 (s, 1H, benzylidene_CH), 5.29 (d, 1H,
J = 3.4, 11.3 Hz, H-3), 4.95–5.07 (m, 2H, H-1 + NH), 4.76 (s, 1H,
Troc_CH2), 4.33–4.42 (m, 2H, H-6a + H-4), 4.05 (dd, 1H, J = 0.8,
12.1 Hz, H-6b), 3.88 (m, 1H, H-2), 3.64 (m, 1H, H-5), 2.05 (s, 3H,
Ac). 13C NMR (100 MHz, CDCl3) dC 170.68 (Ac), 137.48 (Ph),
135.46 (Ph), 129.25 (Ph), 129.06 (Ph), 128.23 (Ph), 126.33 (Ph),
100.83 (PhCH), 96.52 (CCl3), 84.56 (C-1), 74.37 (Troc_CH2), 73.15
(C-4), 71.32 (C-3), 69.74 (C-5), 69.19 (C-6), 50.30 (C-2), 20.85
4.13. p-Chlorophenyl 3-O-acetyl-2-O-benzyl-4,6-O-benzylidene-
1-thio-b-D-galactopyranoside (7)
To a solution of 24 (3.50 g, 7.22 mmol) in pyridine (30 mL), ace-
tic anhydride (20 mL) was added dropwise. The solution was stir-
red at rt for 3 h and then the solvent was evaporated. The
residue was purified by column chromatography on silica gel using
25% EtOAc–hexane as eluent to give the compound 7 (3.62 g, 95.3%
(Ac). HRCIMS: Calcd for
629.0264. Found: 629.0247.
C
24H27N2O7SCl4 (M+NH4þ): m/z
4.11. p-Chlorophenyl 2-O-benzyl-1-thio-b-D-galactopyranoside
(23)
yield). Rf = 0.38 (EtOAc/Hexane 3:7). [
a
]
D
ꢁ5.83 (c 0.96, CHCl3). 1H
NMR (400 MHz, CDCl3) dH 7.68–7.63 (m, 2H, SPh), 7.52–7.28 (m,
10H, benzylidene + Bn), 7.18–7.12 (m, 2H, SPh), 5.52 (s, 1H, PhCH),
4.98 (dd, 1H, J = 9.7, 3.3 Hz, H-3), 4.76 (d, 1H, J = 10.9 Hz, PhCH2),
4.68 (d, 1H, J = 9.5 Hz, H-1), 4.54 (d, 1H, J = 10.9 Hz, PhCH2), 4.43–
4.34 (m, 2H, H_4 + H-6a), 4.04 (dd, 1H, J = 12.4, 1.5 Hz, H-6b),
3.90 (dd, 1H, J = 9.6, 9.6 Hz, H-2), 3.61–3.57 (m, 1H, H-5), 2.02 (s,
3H, Ac). 13C NMR (100 MHz, CDCl3) dC 170.50 (Ac), 138.03 (Ph),
137.66 (Ph), 134.26 (Ph), 133.95 (Ph), 130.71 (Ph), 129.18 (Ph),
129.06 (Ph), 128.36 (Ph), 128.20 (Ph), 127.77 (Ph), 127.63 (Ph),
126.33 (Ph), 100.82 (PhCH), 86.10 (C-1), 75.48 (C-3), 75.30 (PhCH2),
73.77 (C-2), 73.72 (C-4), 69.51 (C-5), 69.13 (C-6), 20.96 (CH3). HRC-
IMS: Calcd for C28H31NO6SCl (M+NH4þ): m/z 544.1561. Found:
544.1570. Anal. Calcd for C28H27O6SCl: C, 63.81; H, 5.16; N, 0.00.
Found: C, 63.42; H, 5.03; N, ꢁ0.06.
Compound 2112 (5.4 g, 17.6 mmol) was dissolved in 2,2-dime-
thoxypropane (30 mL). After adding 10-camphorsulfonic acid
(300 mg), the reaction was refluxed overnight. Et3N was then added
and the solvent was evaporated. The crude was dissolved in anhy-
drous DMF (30 mL) containing NaH (60%, 2.1 g, 52.5 mmol). At 0 °C,
benzyl bromide (2.7 mL, 22.7 mmol) was added and the reaction
was then warmed up to rt. After 1 h, MeOH was added dropwise
and the mixture was extracted with EtOAc–H2O. The organic layer
was dried over anhydrous Na2SO4 and to solvent was evaporated to
obtain crude 22 (6.9 g, crude). Rf = 0.53 (EtOAc/toluene 1:4). The
residue was used for next step without any further purification.
The obtained crude 22 was dissolved in 70% AcOH–H2O (70 mL)
and refluxed for 1 h. The solvent was evaporated and the residue
was washed by CH2Cl2. After filtration, the filtrate was concen-
trated and the residue was purified by column chromatography
on silica gel using 4% MeOH–CH2Cl2 as eluent to give the com-
pound 23 (3.62 g, 51.9% yield over three steps). Rf = 0.32 (MeOH/
4.14. 6-Azidohexyl 2,3,4,6-tetra-O-benzoyl-b-
D-galactopyrano-
syl-(1?4)-2,3,6-tri-O-benzoyl-b- -glucopyranoside (26)
D
CH2Cl2 5:95). [a]
+18.4 (c 1.3, CHCl3). 1H NMR (400 MHz, CDCl3)
Perbenzoylated lactosyl bromide 2528 (20.0 g, 17.6 mmol) and
6-azidohexan-1-ol (4 mL, 26.5 mmol) were dissolved in anhydrous
toluene (35 mL). After adding molecular sieves (3.5 g), the mixture
was stirred for 20 min. At ꢁ78 °C, AgOTf (7.0 g, 29.9 mL) was added
into the mixture and the reaction was kept in darkness, and then
warmed up to rt. After 4 h, Et3N was added and the mixture was fil-
trated. ThesolutionwasthenextractedwithEtOAc/NH3ꢀH2O. Theor-
ganic layer was dried over anhydrous Na2SO4 and the solvent was
evaporated. The residue was purified by column chromatography
on silica gel using 3% EtOAc–toluene as eluent to give 26 as white so-
D
dH 7.53–7.48 (m, 2H, SPh), 7.41–7.32 (m, 5H, Bn), 7.32–7.28 (m,
2H, SPh), 4.95 (d, 1H, J = 11.1 Hz, PhCH2), 4.68 (d, 1H, J = 11.1 Hz,
PhCH2), 4.62 (d, 1H, J = 9.0 Hz, H-1), 4.05 (dd, 1H, J = 2.9, <1 Hz,
H-4), 3.97 (dd, 1H, J = 11.9, 5.9 Hz, H-6a), 3.86 (dd, 1H, J = 11.9,
4.2 Hz, H-6b), 3.67 (dd, 1H, J = 8.8, 3.1 Hz, H-3), 3.63(dd, 1H,
J = 8.9, 8.9 Hz, H-2), 3.54 (m, 1H, H-5). 13C NMR (100 MHz, CDCl3)
dC 137.82 (Ph), 133.80 (Ph), 132.98 (Ph), 132.05 (Ph), 129.15 (Ph),
128.67 (Ph), 128.21 (Ph), 87.37 (C-1), 77.88 (C-5), 77.82 (C-2),
75.35 (PhCH2), 74.81 (C-3), 69.78 (C-4), 62.99 (C-6). HRCIMS: Calcd
for C19H25NO5SCl (M+NH4þ): m/z 414.1142. Found: 414.1126. Anal.
Calcd for C19H21O5SCl: C, 57.50; H, 5.33; N, 0.00. Found: C, 57.54; H,
5.15; N, ꢁ0.04.
lid (19.7 g, 93.6% yield). Rf: 0.84 (EtOAc/toluene 1:4). [a] +25.7
D
(c 1.2, CHCl3). 1H NMR (400 MHz, CDCl3) dH 8.07–7.95 (m, 10H, Bz),
7.95–7.89 (m, 2H, Bz), 7.77–7.72 (m, 2H, Bz), 7.67–7.54 (m, 3H,
Bz), 7.54–7.46 (m, 5H, Bz), 7.46–7.36 (m, 6H, Bz), 7.36–7.29 (m,
3H, Bz), 7.26–7.13 (m, 4H, Bz), 5.82 (dd, 1H, J = 9.7, 9.7 Hz, Glc_H-
3), 5.78–5.71 (m, 2H, Gal_H-2 + Gal_H-4), 5.47 (dd, 1H, J = 9.9,
8.0 Hz, Glc_H-2), 5.39 (dd, 1H, J = 10.3, 3.4 Hz, Gal_H-3), 4.90 (d,
1H, J = 7.9 Hz, Gal_H-1), 4.69 (d, 1H, J = 7.9 Hz, Glc_H-1), 4.62 (dd,
1H, J = 12.1, 1.7 Hz, Glc_H-6a), 4.50 (dd, 1H, J = 12.1, 4.4 Hz,
4.12. p-Chlorophenyl 2-O-benzyl-4,6-O-benzylidene-1-thio-b-D-
galactopyranoside (24)
Compound 23 (3.42 g, 8.62 mmol) was stirred in anhydrous
CH3CN (50 mL). Benzaldehyde dimethyl acetal (2.6 mL, 17.3 mmol)

J. Feng et al. / Carbohydrate Research 346 (2011) 2650–2662
2659
Glc_H-6b), 4.28 (dd, 1H, J = 9.5 Hz, Glc_H-4), 3.91 (ddd, 1H,
J = 6.8, 6.8, <1 Hz, Gal_H-5), 3.88–3.82 (m, 2H, Glc_H-5 + OCH2a),
3.80–3.67 (m, 2H, Gal_H-6a + Gal_H-6b), 3.45 (ddd, 1H, J = 13.1,
7.1, 5.9 Hz, OCH2b), 3.04 (t, 2H, J = 6.9 Hz, CH2N3), 1.57–1.39 (m,
2H, chain), 1.36–1.26 (m, 2H, chain), 1.24–1.10 (m, 4H,
CH2 ꢄ 2_chain). 13C NMR (100 MHz, CDCl3) dC 165.86 (Bz), 165.59
(Bz), 165.42 (Bz ꢄ 2), 165.24 (Bz), 165.14 (Bz), 164.81 (Bz), 133.55
(Ph), 133.41 (Ph), 133.38 (Ph), 133.27 (Ph), 133.23 (Ph), 133.18
(Ph), 130.01 (Ph), 129.76 (Ph), 129.70 (Ph), 129.67 (Ph), 129.60
(Ph), 129.46 (Ph), 129.44 (Ph), 128.89 (Ph), 128.72 (Ph), 128.65
(Ph), 128.59 (Ph), 128.57 (Ph), 128.53 (Ph), 128.40 (Ph), 128.26
(Ph), 101.22 (Glc_ C-1), 101.00 (Gal_ C-1), 76.10 (Glc_ C-4), 73.03
(Glc_ C-5), 72.90 (Glc_ C-3), 71.82 (Glc_ C-2 + Gal_ C-3), 71.43
(Gal_ C-5), 70.01 (OCH2), 69.93 (Gal_ C-4), 67.56 (Gal_ C-2), 62.44
(Glc_ C-6), 61.11 (Gal_ C-6), 51.16 (CH2N3), 29.18 (chain), 28.57
(chain), 26.22 (chain), 25.34 (chain). HRESIMS: Calcd for
60.35 (Glc_C-6), 51.00 (CH2N3), 29.22 (chain), 28.45 (chain), 26.21
(chain), 25.23 (chain). Anal. Calcd for
C, 53.61; H, 6.75; N, 7.50. Found: C, 54.11; H, 6.70; N, 7.01.
C
25H37N3O11ꢀ1/4H2O:
4.17. 6-Azidohexyl 2,3-di-O-benzyl-4,6-O-benzylidene-b-D-
galactopyranosyl-(1?4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside
(29)
Compound 28 (1.0 g, 1.8 mmol) was dissolved in anhydrous
DMF (6 mL), and NaH (480 mg, 10.8 mmol) was added into the
solution. At 0 °C, BnCl (2.1 mL, 18.0 mmol) was added into the mix-
ture. After stirring overnight at rt for 3.5 h, MeOH was added to
quench the reaction. The mixture was diluted by EtOAc, and
washed with water and brine. The organic layer was dried over
anhydrous Na2SO4 and evaporated. The residue was purified by
column chromatography on silica gel using 5% EtOAc–toluene as
eluent to furnish the compound 29 (1.5 g, 83.5% yield). Rf: 0.59
C
67H61N3O18K (M+K+): m/z 1234.3587. Found: 1234.3593. Anal.
Calcd for C67H61N3O18: C, 67.27; H, 5.14; N, 3.51. Found: C, 66.99;
H, 5.33; N, 3.49.
(EtOAc/toluene 0.5:9.5).
[a
]
D
+14.6 (c 0.6, CHCl3). 1H NMR
(400 MHz, CDCl3) dH 7.17–7.58 (m, 30H, PhCH ꢄ 5 + Bn ꢄ 25),
5.49 (s, 1H, PhCH), 5.21 (d, 1H, J = 10.7 Hz, PhCH2), 4.93 (d, 1H,
J = 11.0 Hz, PhCH2), 4.87 (d, 1H, J = 11.2 Hz, PhCH2), 4.73–4.84 (m,
5H, PhCH2 ꢄ 5), 4.58 (d, 1H, J = 12.1 Hz, PhCH2), 4.49 (d, 1H,
J = 7.9 Hz, Gal_H-1), 4.41 (d, 1H, J = 7.9 Hz, Glc_H-1), 4.36 (d, 1H,
J = 12.1 Hz, PhCH2), 4.23 (dd, 1H, J = 1.0, 12.4 Hz, Gal_H-6a), 4.05
(dd, 1H, J = <1, 3.4 Hz, Gal_H-4), 3.83–4.03 (m, 4H, Glc_H-
4 + OCH2a + Glc_H-6a + Gal_H-6b), 3.72–3.82 (m, 2H, Gal_H-
2 + Glc_H-6b), 3.66 (dd, 1H, J = 9.1, 9.1 Hz, Glc_H-3), 3.55 (ddd,
1H, J = 6.7, 9.5, 13.5 Hz, OCH2b), 3.36–3.49 (m, 3H, Glc_H-
2 + Gal_H-3 + Glc_H-5), 3.23 (t, 1H, J = 6.9 Hz, CH2N3), 2.96 (s, 1H,
Gal_H-5), 1.35–1.74 (m, 8H, CH2 ꢄ 8). 13C NMR (100 MHz, CDCl3)
dC 139.05 (Ph), 138.69 (Ph), 138.68 (Ph), 138.37 (Ph), 138.28 (Ph),
138.21 (Ph), 129.11 (Ph), 128.37 (Ph), 128.31 (Ph), 128.28 (Ph),
128.23 (Ph), 128.13 (Ph), 128.03 (Ph), 127.91 (Ph), 127.86 (Ph),
127.77 (Ph), 127.58 (Ph), 127.54 (Ph), 127.51 (Ph), 127.42 (Ph),
127.28 (Ph), 126.27 (Ph), 103.65 (Glc_C-1), 103.24 (PhCH), 101.79
(Gal_C-1), 82.96 (Glc_C-2), 81.87 (Gal_C-5), 80.27 (Gal_C-4),
77.95 (Gal_C-3), 76.71 (Gal_C-2), 75.33 (PhCH2), 75.07 (Glc_C-5),
74.89 (PhCH2), 73.80 (Glc_C-4), 73.41 (PhCH2), 73.38 (PhCH2),
73.23 (PhCH2), 72.22 (Glc_C-3), 69.73 (OCH2), 69.03(Gal_C-6),
68.28 (Glc_C-6), 51.36 (CH2N3), 29.61 (chain), 28.77 (chain),
26.52 (chain), 25.74 (chain). HRESIMS: Calcd for C60H67N3O11Na
(M+Na+): m/z 1028.4668. Found: 1028.4677.
4.15. 6-Azidohexyl b-
glucopyranoside (27)
D-galactopyranosyl-(1?4)-b-D-
A solution of 26 (19.7 g, 16.5 mmol) in anhydrous MeOH
(200 mL) was refluxed at 60 °C in the presence of NaOMe (2.0 mL,
1.5 M in MeOH) for 3 h. Amberlite IR-120 resin (H+) was then added
into the solution to neutralize the base. The mixture was filtrated
and the solvent was evaporated. The solid was washed with acetone
and filtrated to produce 27 as white solid (7.43 g, 96.5% yield). Rf:
0.71 (MeOH/CH2Cl2 1:4). [
a]
ꢁ4.2 (c 1.0, MeOH). 1H NMR
D
(400 MHz, CD3OD) dH 4.37 (d, 1H, J = 7.8 Hz, Gal_ H-1), 4.28 (d,
1H, J = 7.8 Hz, Glc_ H-1), 3.95–3.85 (m, 2H, OCH2a + Glc_H-6a),
3.85–3.67 (m, 4H, Glc_H-6b + Gal_ H-4 + Gal_H-6a + Gal_H-6b),
3.63–3.47 (m, 6H, Gal_ H-5 + Gal_ H-2 + OCH2b + Gal_ H-3 + Glc_
H-3 + Glc_ H-4), 3.40 (ddd, 1H, J = 9.2, 3.9, 2.6 Hz, Glc_ H-5), 3.29
(t, 2H, J = 6.9 Hz, CH2N3), 3.25 (dd, 1H, J = 8.0, 7.3 Hz, Glc_ H-2),
1.70–1.36 (m, 8H, chain). 13C NMR (100 MHz, D2O) dC 102.93
(Gal_C-1), 102.02 (Glc_C-1), 78.43 (Gal_C-2), 75.35 (Gal_C-5),
74.75 (Glc_C-3), 74.46 (Glc_C-5), 72.85 (Glc_C-4), 72.53 (Glc_C-2),
70.96 (Gal_C-3), 70.54 (OCH2), 68.55 (Gal_C-4), 61.02 (Gal_C-6),
60.12 (Glc_C-6), 51.14 (CH2N3), 28.58 (chain), 27.87 (chain), 25.65
(chain), 24.60 (chain). Anal. Calcd for C18H33N3O11: C, 46.25; H,
7.12; N, 8.99. Found: C, 46.02; H, 7.11; N, 8.81.
4.18. 6-Azidohexyl 2,3,6-tri-O-benzyl-b-
D-galactopyranosyl-
4.16. 6-Azidohexyl 4,6-O-benzylidene-b-
D
-galactopyranosyl-
(1?4)-2,3,6-tri-O-benzyl-b- -glucopyranoside (8)
D
(1?4)-b- -glucopyranoside (28)
D
Compound 29 (1.24 g, 1.23 mmol) was dissolved in anhydrous
CH2Cl2 (50 mL). At 0 °C, Et3SiH (2.0 mL, 12.3 mmol) was added
To a solution of 27 (3.0 g, 6.42 mmol) in anhydrous DMF (20 mL),
PhCH(OMe)2 (3 mL, 19.3 mmol) and 10-camphorsulfonic acid
(550 mg, 2.37 mmol) were added. After stirring for 1 h at rt, Et3N
was used to neutralize the acid. The solvent was evaporated and
the residue was purified by column chromatography on silica gel
using 5% MeOH–CH2Cl2 as eluent to yield the compound 28 (2.8 g,
and then BF3ꢀEt2O (232.4
lL, 1.85 mmol) was added dropwise into
the mixture. The reaction was stirred overnight keeping the tem-
perature at 0 °C overnight. Et3N was then added to quench the
reaction. The solvent was evaporated and the residue was purified
by column chromatography on silica gel using 15% EtOAc–hexane
as eluent to give the compound 8 as a colorless gel (690 mg, 55.6%
78.6% yield). Rf: 0.36 (MeOH/CH2Cl2 1:9). [
a]
D ꢁ27.1 (c 1.0, MeOH).
1H NMR (400 MHz, CD3OD) dH 7.58–7.30 (m, 5H, Ph), 5.63 (s, 1H,
PhCH), 4.49 (d, 1H, J = 7.3 Hz, Gal_H-1), 4.30 (d, 1H, J = 7.9 Hz,
Glc_H-1), 4.25–4.18 (m, 2H, Gal_H-4 + Gal_H-6a), 4.16 (dd, 1H,
J = 1.5, 12.5 Hz, Gal_H-6b), 3.97–3.86 (m, 3H, Glc_H-6a + Glc_H-
6b + OCH2a), 3.73–3.52 (m, 6H, Gal_H-2 + Gal_H-3 + Gal_H-
5 + Glc_H-3 + Glc_H-4 + OCH2b), 3.41 (ddd, 1H, J = 2.7, 2.7, 9.3 Hz,
Glc_H-5), 3.30–3.23 (m, 2H, CH2N3 + Glc_H-2), 1.70–1.55 (m, 4H,
chain), 1.50–1.36 (m, 4H, chain). 13C NMR (100 MHz, CD3OD) dC
138.15 (Ph), 128.49 (Ph), 127.63 (Ph), 126.10 (Ph), 103.43 (Gal_C-1),
102.87 (Glc_C-1), 100.88 (PhCH), 78.66 (Glc_C-3), 75.95 (Gal_C-4),
75.04 (Glc_C-5), 74.94 (Glc_C-4), 73.43 (Glc_C-2), 72.10 (Gal_C-3),
70.36 (Gal_C-2), 69.36 (OCH2), 68.79 (Gal_C-6), 66.92 (Gal_C-5),
yield). Rf: 0.67 (EtOAc/toluene 1:3). [a]
+13.3 (c 3.0, CHCl3). 1H
D
NMR (400 MHz, CDCl3) dH 7.42–7.19 (m, 30H, Bn ꢄ 30), 4.99 (d,
1H, J = 10.9 Hz, PhCH2), 4.89 (d, 1H, J = 11.0 Hz, PhCH2), 4.80–4.70
(m, 5H, PhCH2 ꢄ 5), 4.67 (d, 1H, J = 11.7 Hz, PhCH2), 4.56 (d, 1H,
J = 12.1 Hz, PhCH2), 4.46 (d, 1H, J = 12.0 Hz, PhCH2), 4.44 (d, 1H,
J = 7.8 Hz, Gal_H-1), 4.43–4.39 (m, 2H, PhCH2 ꢄ 2), 4.38 (d, 1H,
J = 7.9 Hz, Glc_H-1), 4.03 (dd, 1H, J = 2.5, 2.5 Hz, Gal_H-4), 4.00–
3.90 (m, 2H, Glc_H-3 + OCH2a), 3.81 (dd, 1H, J = 4.5, 11.0 Hz,
Gal_H-6a), 3.73 (dd, 1H, J = 1.7, 10.9 Hz, Gal_H-6b), 3.67 (dd, 1H,
J = 7.2, 9.7 Hz, Glc_H-6a), 3.63–3.46 (m, 4H, Gal_H-2 + Glc_H-
4 + OCH2b + Glc_H-6b), 3.44–3.36 (m, 3H, Glc_H-2 + Gal_H-
3 + Gal_H-5), 3.33 (m, 1H, Glc_H-5), 3.23 (t, 1H, J = 7.0 Hz,

2660
J. Feng et al. / Carbohydrate Research 346 (2011) 2650–2662
CH2N3), 2.40 (d, 1H, J = 2.1 Hz, Gal_OH), 1.70–1.35 (m, 8H,
CH2 ꢄ 8). 13C NMR (100 MHz, CDCl3) dC 139.17 (Ph), 138.75 (Ph),
138.67 (Ph), 138.38 (Ph), 138.23 (Ph), 137.96 (Ph), 128.47 (Ph),
128.37 (Ph), 128.27 (Ph), 128.07 (Ph), 128.06 (Ph), 127.90 (Ph),
127.86 (Ph), 127.80 (Ph), 127.77 (Ph), 127.66 (Ph), 127.61 (Ph),
127.53 (Ph), 127.47 (Ph), 127.23 (Ph), 103.63 (Glc_C-1), 102.56
(Gal_C-1), 82.93 (Glc_C-4), 81.84 (Glc_C-2), 81.16 (Gal_C-5),
79.43 (Gal_C-2), 76.67 (Glc_C-3), 75.34 (PhCH2), 75.25 (PhCH2),
75.15 (Gal_C-3), 74.89 (PhCH2), 73.52 (PhCH2), 73.14 (PhCH2),
72.80 (Glc_C-5), 72.03 (PhCH2), 69.73 (OCH2), 68.46 (Glc_C-6),
68.36 (Gal_C-6), 66.17 (Gal_C-4), 51.37 (CH2N3), 29.62 (chain),
28.79 (chain), 26.54 (chain), 25.75 (chain). HRESIMS: Calcd for
(3 mL). The reaction was stirred at rt for 2 h. The mixture was
then neutralized by Amberlite IR-120 resin (H+). After filtration,
the solvent was evaporated. The residue was purified by column
chromatography on silica gel using 20% EtOAc–hexane as eluent
to afford the compound 6 as a colorless gel. (318.2 mg, 96.5%
yield). Rf: 0.56 (EtOAc/toluene 1:4). [
1H NMR (400 MHz, CDCl3) d 7.48–7.21 (m, 40H, Ph), 5.42 (s, 1H,
PhCH), 5.23 (d, 1H, J = 3.2 Hz, Gal_H-1), 5.11 (d, 1H, J = 11.7 Hz,
a]
+48.2 (c 0.86, CHCl3).
D
a
Bn), 4.95–4.88 (m, 2H, Bn), 4.87–4.70 (m, 6H, Bn), 4.67 (d, 1H,
J = 11.5 Hz, Bn), 4.61 (d, 1H, J = 12.1 Hz, Bn), 4.54 (d, 1H,
J = 7.6 Hz, bGal_H-1), 4.47–4.40 (m, 2H, Bn + Glc_H-1), 4.36 (d,
1H, J = 11.8 Hz, Bn), 4.30 (d, 1H, J = 11.8 Hz, Bn), 4.22 (dd, 1H,
C
60H69N3O11Na (M+Na+): m/z 1030.4824. Found: 1030.4866. Anal.
J = 9.5, <1 Hz,
4 + bGal_H-6a + bGal_H-5), 4.06–3.93 (m, 2H, Glc_H-3 + OCH2),
3.92–3.85 (m, 2H, Gal_H-2 + Glc_H-6a), 3.80 (dd, 1H, J = 10.7,
1.5 Hz, Glc_H-6b), 3.70–3.52 (m, 5H, Glc_H-4 + bGal_H-2 +
Gal_H-6a + Gal_H-6b + OCH2), 3.51–3.42 (m, 3H, bGal_H-6b +
Glc_H-5 + Glc_H-2), 3.40–3.32 (m, 2H, Gal_H-5 + bGal_H-3),
aGal_H-3), 4.18–4.09 (m, 4H, bGal_H-4 + aGal_H-
Calcd for C60H69N3O11: C, 71.48; H, 6.90; N, 4.17. Found: C,
71.24; H, 6.79; N, 4.64.
a
4.19. 6-Azidohexyl 3-O-acetyl-2-O-benzyl-4,6-O-benzylidene-
-galactopyranosyl-(1?4)-2,3,6-tri-O-benzyl-b- -galactopyrano-
syl-(1?4)-2,3,6-tri-O-benzyl-b- -glucopyranoside (30)
a-
a
a
D
D
a
D
3.25 (t, 2H, J = 6.9 Hz, N3CH2), 2.18 (s, br, 1H, OH), 1.75–1.65
(m, 2H, chain), 1.65–1.55 (m, 2H, chain), 1.50–1.38 (m, 4H, chain).
HRESIMS: Calcd for C80H89N3O16Na (M+Na+): m/z 1370.6135.
Found: 1370.6115.
Compound 7 (235.2 mg, 0.45 mmol) and 8 (300 mg, 0.30 mmol)
were dissolved in anhydrous CH2Cl2 (3 mL) containing molecular
sieves (200 mg). The mixture was stirred at rt for 15 min and then
cooled down to ꢁ78 °C. N-Iodosuccinimide (133.9 mg, 0.60 mmol)
and trifluoromethanesulfonic acid was added and the reaction was
slowly warmed up to rt. Et3N was then added and the residue was
purified by column chromatography on silica gel using 15% EtOAc–
hexane as eluent to yield the compound 30 (362.5 mg, 86.9% yield).
4.21. 6-Azidohexyl 3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-N-
(2,2,2-trichloroethoxycarbonylamino)-b-
(1?3)-2-O-benzyl-4,6-O-benzylidene-
(1?4)-2,3,6-tri-O-benzyl-b- -galactopyranosyl-(1?4)-2,3,6-tri-
O-benzyl-b- -glucopyranoside (31)
D
-galactopyranosyl-
a
-D-galactopyranosyl-
D
Rf: 0.34 (EtOAc/toluene 1:9, developed twice). [
CHCl3). 1H NMR (400 MHz, CDCl3) dH 7.45–7.14 (m, 40H, Ph), 5.33
(s, 1H, PhCH), 5.28 (dd, 1H, J = 10.7, 3.4 Hz, Gal_H-3), 5.18 (d, 1H,
J = 3.3 Hz, Gal_H-1), 5.04 (d, 1H, J = 11.8 Hz, PhCH2), 4.94–4.80
a
]
D
+60.0 (c 0.99,
D
a
Compound
5
(235.7 mg, 0.38 mmol) and
6
(260 mg,
a
0.19 mmol) were dissolved in anhydrous CH2Cl2 (3 mL) contain-
ing molecular sieves (120 mg). The mixture was stirred at rt for
30 min. At ꢁ78 °C, N-iodosuccinimide (108.4 mg, 0.38 mmol)
and trifluoromethanesulfonic acid were added into the mixture.
The reaction was slowly warmed up to ꢁ20 °C for 1 h. Et3N was
then added to quench the reaction. The mixture was purified by
column chromatography on silica gel using 22% EtOAc–hexane
as eluent to furnish the compound 31 (330 mg, 95.8% yield). Rf:
(m, 3H, PhCH2), 4.80–4.67 (m, 3H, PhCH2), 4.65–4.52 (m, 4H, PhCH2),
4.48 (d, 1H, J = 7.6 Hz, bGal_H-1), 4.42–4.34 (m, 3H, PhCH2 + a-
Gal_H-4 + Glc_H-1), 4.34–4.22 (m, 2H, PhCH2), 4.19–4.06 (m, 4H,
aGal_H-2 + aGal_H-5 + bGal_H-6a + bGal_H-4), 3.99–3.88 (m, 2H,
Glc_H-3 + OCH2), 3.83 (dd, 1H, J = 10.8, 4.3 Hz, Glc_H-6a), 3.75 (dd,
1H, J = 9.3, 1.7 Hz, Glc_H-6b), 3.66 (dd, 1H, J = 9.9, 7.7 Hz, bGal_H-
2), 3.60 (dd, 1H, J = 9.0, 9.0 Hz, Glc_H-4), 3.56–3.42 (m, 4H,
OCH2 +
aGal_H-6a + bGal_H-6b +
aGal_H-6b), 3.42–3.26 (m, 4H,
0.52 (EtOAc/toluene 1:4). [
(400 MHz, CDCl3) dH 7.56–7.14 (m, 45H, Ph), 5.44 (s, 1H, PhCH),
5.42 (s, 1H, PhCH), 5.21 (d, 1H, J <1 Hz, Gal_H-1), 5.06 (d, 1H,
a]
+47.8 (c 4.0, CHCl3). 1H NMR
D
Glc_H-2 + bGal_H-5 + Glc_H-5 + bGal_H-3), 3.21 (t, 2H, J = 6.9 Hz,
CH2N3), 1.71–1.50 (m, 4H, chain_CH2), 1.46–1.32 (m, 4H,
chain_CH2). 13C NMR (100 MHz, CDCl3) dC 170.55 (Ac), 139.46 (Ph),
138.76 (Ph), 138.46 (Ph), 138.20 (Ph), 137.92 (Ph), 128.77 (Ph),
128.41 (Ph), 128.38 (Ph), 128.26 (Ph), 128.21 (Ph), 128.18 (Ph),
128.14 (Ph), 128.00 (Ph), 127.98 (Ph), 127.94 (Ph), 127.86 (Ph),
127.62 (Ph), 127.59 (Ph), 127.55 (Ph), 127.53 (Ph), 127.50 (Ph),
127.40 (Ph), 127.05 (Ph), 126.89 (Ph), 126.16 (Ph), 103.55 (Glc_C-
a
J = 11.1 Hz, Bn), 4.93–4.77 (m, 5H, Bn), 4.77–4.64 (m, 3H, Bn),
4.64–4.56 (m, 2H, Bn), 4.56–4.49 (m, 2H, bGal_H-1 + Bn), 4.49–
4.40 (m, 2H, Bn + Troc), 4.40–4.34 (m, 3H,
1 + GalN_H-1), 4.34–4.11 (m, 8H,
6a + Gal_H-3 + Gal_H-2 + GalN_H-6a), 4.11–4.01 (m, 3H,
Gal_H-5 + Glc_H-3 + bGal_H-4), 4.01–3.91 (m, 2H, GalN_H-
4 + OCH2), 3.91–3.73 (m, 3H, Glc_H-6a + GalN_H-6b + Glc_H-6b),
3.68–3.49 (m, 6H, bGal_H-2 + Gal_H-6a + Gal_H-6b + Glc_H-
a
Gal_H-4 + Glc_H-
Bn + Troc + bGal_H-
a
a
a
1), 103.01 (bGal_C-1), 100.46 (aGal_C-1), 100.39 (PhCH), 82.56
(Glc_C-4), 81.71 (bGal_C-5), 81.19 (bGal_C-3), 78.74 (bGal_C-2),
a
a
77.43 (Glc_C-3), 75.08 (PhCH2), 75.03 (Glc_C-2), 74.98 (PhCH2),
4 + bGal_H-6b + OCH2), 3.42–3.29 (m, 4H, Glc_H-2 + Glc_H-
5 + bGal_H-3 + bGal_H-5), 3.23 (t, 2H, J = 6.9 Hz, N3CH2), 2.67–
2.56 (m, 1H, GalN_H-5), 2.02 (s, 3H, Ac), 1.71–1.52 (m, 4H, chain),
1.50–1.34 (m, 4H, chain). 13C NMR (100 MHz, CDCl3) d 170.69,
153.87, 139.28, 138.62, 138.43, 138.34, 138.30, 138.13, 138.05,
138.03, 137.61, 129.51, 128.93, 128.64, 128.60, 128.55, 128.40,
128.38, 128.27, 128.22, 128.11, 127.96, 127.90, 127.86, 127.80,
127.69, 127.65, 127.61, 127.57, 127.50, 127.47, 127.15, 126.36,
126.28, 126.21, 103.60, 102.63, 101.71, 100.74, 100.51, 100.24,
95.58, 81.89, 81.53, 81.16, 78.85, 76.85, 76.36, 75.06, 74.87,
74.80, 74.65, 74.47, 74.06, 73.46, 73.06, 72.77, 72.66, 72.01,
71.13, 69.69, 68.99, 68.27, 67.06, 65.58, 63.24, 51.91, 51.31,
50.71, 47.94, 29.88, 29.56, 28.72, 26.47, 25.69, 20.80. HRESIMS:
Calcd for C98H107N4O23Cl3Na (M+Na+): m/z 1837.6292. Found:
1837.6323.
74.83 (PhCH2), 74.21 (
73.66 ( Gal_C-5) , 73.09 (PhCH2), 73.04 (PhCH2), 72.80 (Glc_C-5),
72.16 (PhCH2), 71.73 ( Gal_C-3), 69.70 (OCH2), 68.97 ( Gal_C-6),
aGal_C-4), 74.05 (aGal_C-2), 73.73 (PhCH2),
a
a
a
68.39 (Glc_C-6), 67.10 (bGal_C-6), 62.36 (bGal_C-4), 51.34
(CH2N3), 29.58 (chain), 28.75 (chain), 26.50 (chain), 25.72 (chain),
21.04 (Ac). HRESIMS: Calcd for C82H91N3O17Na (M+Na+): m/z
1412.6241. Found: 1412.6221.
4.20. 6-Azidohexyl 2-O-benzyl-4,6-O-benzylidene-
pyranosyl-(1?4)-2,3,6-tri-O-benzyl-b- -galactopyranosyl-
(1?4)-2,3,6-tri-O-benzyl-b- -glucopyranoside (6)
a-D-galacto-
D
D
NaOMe (100
lL, 1.5 M in MeOH) was added to a solution of 30
(340 mg, 0.24 mmol) in a mixture of CH2Cl2 (1 mL) and MeOH

J. Feng et al. / Carbohydrate Research 346 (2011) 2650–2662
2661
4.22. 6-Azidohexyl 4,6-O-benzylidene-2-deoxy-2-N-
methoxycarbonylamino-b- -galactopyranosyl-(1?3)-2-O-
benzyl-4,6-O-benzylidene- -galactopyranosyl-(1?4)-2,3,6-
tri-O-benzyl-b- -galactopyranosyl-(1?4)-2,3,6-tri-O-benzyl-b-
-glucopyranoside (32)
6.9 Hz, H-6a), 4.11 (dd, 1H, J = 11.3, 6.2 Hz, H-6b), 3.94–3.86 (m,
1H, H-5), 3.63 (dd, 1H, J = 10.2, 10.2 Hz, H-2), 2.11 (s, 3H, Ac),
2.06 (s, 3H, Ac), 2.05 (s, 3H, Ac). 13C NMR (100 MHz, CDCl3) dC
170.32 (Ac), 169.84 (Ac), 169.66 (Ac), 135.10 (Ph), 135.04 (Ph),
129.34 (Ph), 129.16 (Ph), 86.25 (C-1), 74.52 (C-5), 72.89 (C-3),
66.49 (C-4), 61.53 (C-6), 59.29 (C-2), 20.64 (CH3), 20.57 (CH3),
20.53 (CH3). HRESIMS: Calcd for C18H20N3O7SClNa (M+Na+): m/z
480.0603. Found: 480.0597.
D
a
-D
D
D
Compound 31 (240 mg, 0.13 mmol) was dissolved in a mixture
of CH2Cl2 (0.7 mL) and MeOH (2.0 mL). NaOMe (100 L, 1.5 M in
l
MeOH) was added into the mixture. Then the reaction was stirred
at rt for 2 h. The mixture was then neutralized by Amberlite IR-120
resin (H+). After filtration, the solution was concentrated. The res-
idue was purified by column chromatography on silica gel using
40% EtOAc–hexane as eluent to give compound 32 (102 mg,
4.25. 3,4,6-Tri-O-acetyl-2-azido-2-deoxy-a,b-D-galactopyranosyl
2,2,2-trichloroacetimidate (11)
Thioglycoside 35 (745 mg, 1.62 mmol) was dissolved in a mix-
ture of CH3CN (5 mL)–H2O (0.6 mL) and treated with N-iodosuccin-
imide (1.46 g, 6.92 mmol) by following a previous procedure12 to
afford the hemiacetal 36 as a crude material. Part of the crude
material (ꢃ200 mg) was converted to the previously known imi-
date 11 by reacting with CCl3CN (0.3 mL) and K2CO3 (166 mg) in
anhydrous CH2Cl2 (1.5 mL) as before.25
46.5% yield). Rf: 0.17 (EtOAc/toluene 1:3). [a]D +43.1 (c 1.8, CHCl3).
1H NMR (400 MHz, CDCl3) dH 7.52–7.15 (m, 45H), 5.45 (s, 1H), 5.44
(s, 1H), 5.23 (d, 1H, J <1), 5.01 (d, 1H, J = 10.9 Hz), 4.89–4.72 (m,
6H), 4.72–4.63 (m, 2H), 4.63–4.55 (m, 2H), 4.51 (d, 1H,
J = 7.6 Hz), 4.41 (d, 1H, J = 12.1 Hz), 4.38–4.19 (m, 6H), 4.17–4.08
(m, 4H), 4.06–3.98 (m, 2H), 3.94 (ddd, 1H, J = 9.5, 9.5, 6.3 Hz),
3.89–3.79 (m, 3H), 3.78–3.67 (m, 2H), 3.66–3.49 (m, 6H), 3.43–
3.28 (m, 4H), 3.23 (t, 2H, J = 6.9 Hz,), 3.09 (s, 1H), 2.61–2.54 (m,
1H), 1.70–1.52 (m, 4H), 1.47–1.34 (m, 4H). 13C NMR (100 MHz,
CDCl3) dC 157.96, 139.15, 138.67, 138.44, 138.33, 138.13, 138.10,
138.07, 137.62, 129.39, 129.06, 128.52, 128.50, 128.44, 128.41,
128.26, 128.18, 127.93, 127.88, 127.80, 127.74, 127.69, 127.66,
127.61, 127.58, 127.53, 127.51, 127.48, 127.22, 126.36, 126.32,
103.64, 102.74, 102.05, 101.12, 100.42, 100.38, 82.03, 81.62,
81.21, 78.84, 76.42, 75.11, 74.93, 74.81, 74.71, 74.69, 74.62,
73.18, 73.11, 72.80, 72.07, 69.73, 69.11, 69.03, 68.31, 67.13,
66.01, 63.33, 55.00, 52.20, 51.35, 29.59, 28.76, 26.51, 25.73. HRE-
SIMS: Calcd for C95H106N4O22Na (M+Na+): m/z 1677.7191. Found:
1677.7157.
4.26. p-Chlorophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-
a-D-
galactopyranosyl-(1?3)-4,6-O-benzylidene-2-deoxy-2-N-(2,2,2-
trichloroethoxycarbonylamino)-1-thio-b-D-galactopyranoside
(37)
Compound 11 (83.6 mg, 0.18 mmol) and 12 (50 mg, 0.09 mmol)
were dissolved in anhydrous CH2Cl2 (1 mL) containing molecular
sieves. The mixture was stirred at rt for 15 min. At ꢁ78 °C, trimeth-
ylsilyl trifluoromethanesulfonate was added and the reaction was
warmed up to 0 °C. Et3N was then added and the mixture was puri-
fied by column chromatography on silica gel using 1% acetone–
CH2Cl2 as eluent to give the
for ). Rf: 0.71 (EtOAc/CH2Cl2 1:4). [a]
D
a
anomer 37 (45.6 mg, 57.4% yield
a
+36.7 (c 0.77, CHCl3). 1H
4.23. p-Chlorophenyl 2-azido-2-deoxy-1-thio-b-
galactopyranoside (34)
D
-
NMR (400 MHz, CDCl3) dH 7.65–7.57 (m, 2H, SPhCl), 7.49–7.36
(m, 5H, benzylidene), 7.21–7.13 (m, 2H, SPhCl), 5.66–5.56 (m, 2H,
NH + PhCH), 5.39 (d, 1H, J = 2.2 Hz, H-40), 5.31–5.23 (m, 2H, H-
30 + H-1), 5.17 (d, 1H, J = 3.4 Hz, H-10), 4.86 (d, 1H, J = 12.0 Hz,
Troc_CH2), 4.64 (d, 1H, J = 12.0 Hz, Troc_CH2), 4.52 (d, 1H,
J = 9.9 Hz, H-3), 4.45–4.37 (m, 2H, H-6a + H-4), 4.21 (dd, 1H,
J = 6.6 Hz, H-50), 4.15–4.00 (m, 3H, H-60a + H-6b + H-60b), 3.65–
3.53 (m, 3H, H-5 + H-20 + H-2), 2.14 (s, 3H, Ac), 2.09 (s, 3H, Ac),
2.02 (s, 3H, Ac). 13C NMR (100 MHz, CDCl3) dC 169.91 (Ac ꢄ 2),
169.57 (Ac), 153.75 (Troc), 137.45 (Ph), 135.02 (Ph), 134.59 (Ph),
133.54 (Ph), 129.17 (Ph), 129.06 (Ph), 128.12 (Ph), 126.17 (Ph),
100.71 (PhCH), 95.86 (C-10). 95.52 (CCl3), 83.50 (C-1), 74.55
(Troc_CH2), 74.45 (C-3), 71.89 (C-4), 69.74 (C-5), 69.32 (C-6),
67.53 (C-30), 67.23 (C-40), 66.97 (C-50), 61.36 (C-60), 56.97 (C-2),
51.44 (C-20), 20.83 (Ac), 20.56 (Ac ꢄ 2). HRESIMS: Calcd for
Sodium azide (306 mg, 4.71 mmol) was suspended in anhy-
drous pyridine (3 mL). At 0 °C, trifluoromethanesulfonic anhydride
(659 lL, 3.92 mmol) was added dropwise. After 2 h, at 0 °C, the
solution was carefully added into an anhydrous pyridine solution
(2.5 mL) containing 19 (1.0 g, 3.27 mmol), Et3N (1.14 mL.
8.18 mmol) and CuSO4 (5.2 mg, 0.03 mmol). The reaction was then
warmed up to rt and stirred for 5 h. The solution was concentrated
to produce crude 34 and the residue used for the next step without
further purification. Rf = 0.48 (MeOH/CH2Cl2 8:92). 1H NMR
(400 MHz, CD3OD) dH 7.60–7.54 (m, 2H, Ph), 7.36–7.30 (m, 2H,
Ph), 4.52 (d, 1H, J = 9.8 Hz, H-1), 3.86 (dd, 1H, J = 2.7, <1 Hz, H-4),
3.78 (dd, 1H, J = 11.5, 6.9 Hz, H-6a), 3.71 (dd, 1H, J = 11.5, 5.1 Hz,
H-6b), 3.57–3.47 (m, 3H, H-5 + H-3 + H-2). 13C NMR (100 MHz,
CD3OD) dC 133.41 (Ph), 133.24 (Ph), 131.77 (Ph), 128.61 (Ph),
86.30 (H-1), 79.36 (H-5), 73.80 (H-3), 68.30 (H-4), 62.96 (H-2),
61.24 (H-6). HRESIMS: Calcd for C12H14N3O4SClNa (M+Na+): m/z
354.0286. Found: 354.0284.
C
34H36N4O13SCl4Na (M+Na+): m/z 903.0646. Found: 903.0664.
4.27. p-Chlorophenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
a-
D-galactopyranosyl-(1?3)-4,6-O-benzylidene-2-deoxy-2-N-
(2,2,2-trichloroethoxycarbonylamino)-1-thio-b-D-
galactopyranoside (10)
4.24. p-Chlorophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-
b-
D
-galactopyranoside (35)
Compound 37 (30 mg, 0.034 mmol) was dissolved in thioacetic
acid (2 mL). The solution was stirred at rt for 15 h and then diluted
by toluene and evaporated. The residue was purified by column
chromatography on silica gel using 65% EtOAc–hexane as eluent
to give the compound 10 (30.0 mg, 98.2% yield). Rf: 0.46 (EtOAc/
Acetic anhydride (5 mL) was added to a solution of 34 (ꢃ1.2 g,
crude) in pyridine (7 mL) and the reaction was stirred at rt over-
night. The solution was then evaporated and the residue was puri-
fied by column chromatography on silica gel using 15% EtOAc–
hexane as eluent to give the compound 35 (1.23 g, 82.3% yield over
hexane 9:1). [a]
+55.7 (c 0.43, CHCl3). 1H NMR (400 MHz, CDCl3)
D
dH 7.69–7.63 (m, 2H, Ph), 7.42–7.37 (m, 3H, Ph), 7.33–7.28 (m,
4H, Ph), 5.55 (d, 1H, J = 9.5 Hz, NH), 5.45 (s, 1H, PhCH), 5.38 (d,
1H, J = 8.8 Hz, NH), 5.31 (dd, 1H, J = 2.2, <1 Hz, H-40), 5.05 (d, 1H,
J = 3.7 Hz, H-10), 4.96–4.87 (m, 3H, H-30 + Troc_CH2 + H-1), 4.69
(d, J = 12.1 Hz, 1H, Troc_CH2), 4.53 (ddd, 1H, J = 11.2, 9.8, 3.7 Hz,
two steps). Rf = 0.75 (EtOAc/Hexane 4:6). [a] +13.5 (c 1.2, CHCl3).
D
1H NMR (400 MHz, CDCl3) dH 7.60–7.54 (m, 2H, Ph), 7.37–7.32 (m,
2H, Ph), 5.37 (dd, 1H, J = 3.2, <1 Hz, H-4), 4.88 (dd, 1H, J = 10.2,
3.2 Hz, H-3), 4.50 (d, 1H, J = 10.1 Hz, H-1), 4.18 (dd, 1H, J = 11.4,

2662
J. Feng et al. / Carbohydrate Research 346 (2011) 2650–2662
H-20), 4.38 (dd, 1H, J = 12.4, 1.3 Hz, H-6a), 4.30 (dd, 1H, J = 3.0,
<1 Hz, H-4), 4.18 (dd, 1H, J = 6.7, 6.7 Hz, H-3), 4.11–4.02 (m, 4H,
H-50 + H-60a + H-60b + H-6b), 3.83 (ddd, 1H, J = 19.4, 9.9, 2.4 Hz,
H-2), 3.61–3.56 (m, 1H, H-5), 2.15 (s, 3H, OAc), 2.10 (s, 3H, OAc),
1.91 (s, 3H, OAc), 1.26 (s, 3H, NAc). 13C NMR (100 MHz, CDCl3) dC
171.00 (Ac), 170.46 (Ac), 170.43 (Ac), 170.23 (Ac), 137.31 (Ph),
136.16 (Ph), 135.10 (Ph), 129.65 (Ph), 129.15 (Ph), 128.43 (Ph),
128.37 (Ph), 126.44 (Ph), 101.34 (PhCH), 95.34 (CCl3), 93.01 (C-
10), 84.08 (C-1), 74.69 (Troc_CH2), 73.19 (C-50), 70.22 (C-4), 69.57
(C-5), 69.47 (C-6), 68.39 (C-30), 66.93 (C-3), 66.89 (C-40), 61.80
(C-60), 50.67 (C-2), 46.82 (C-20), 22.33 (NAc), 20.91 (OAc), 20.67
1.5 M in MeOH) was added. The reaction was stirred at 50 °C for
2 h and was concentrated. The residue was mixed in aq. NaOH
(2 N, 4 mL) and the mixture was kept at 100 °C to reflux overnight.
After evaporating the solvent, the residue was stirred in a mixture
of Ac2O (2 mL)/pyridine (3 mL) and followed by the treatment of
MeOH (4 mL)/NaOMe (100 lL, 1.5 M in MeOH). The mixture was
concentrated and the residue was purified by column chromatog-
raphy on silica gel using 2% MeOH–CH2Cl2 as eluent to afford the
intermediate 38, which was then dissolved in a mixture of MeOH
(5 mL), H2O (50 lL) and several drops of AcOH, and was treated
by Pd(OH)2/C/H2 at rt for 2 days. Then the Pd(OH)2/C was filtered
and the solution was concentrated to afford the compound 2
(2 mg, 10% yield). Rf: 0.25 (IPA/H2O/NH3ꢀH2O 6:2:1). Selected 1H
(OAc), 20.60 (OAc). HRESIMS: Calcd for
C36H40N2O14SCl4Na
(M+Na+): m/z 919.0847. Found: 919.0862.
NMR (400 MHz, D2O) dH 5.10 (d, 1H, J = 3.7 Hz, H-1_aGalNAc),
4.28. 6-Azidohexyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
galactopyranosyl-(1?3)-4,6-O-benzylidene-2-deoxy-2-N-(2,2,2-
trichloroethoxycarbonylamino)-b- -galactopyranosyl-(1?3)-2-
O-benzyl-4,6-O-benzylidene- -galactopyranosyl-(1?4)-2,3,6-
tri-O-benzyl-b- -galactopyranosyl-(1?4)-2,3,6-tri-O-benzyl-b-
-glucopyranoside (9)
a
-
D
-
4.96 (d, 1H, J = 4.0 Hz, H-1_
H-1_bGalNAc), 4.54 (d, 1H, J = 7.8 Hz, H-1_bGal), 4.51 (d, 1H,
J = 8.2 Hz, H-1_
GalNAc), 3.53 (t, 2H, J = 5.1 Hz, CH2NH3þ), 2.09
aGal), 4.74 (overlaped with HDO, 1H,
D
a
a
-D
(s, 3H, NHAc), 2.07 (s, 3H, NHAc). RESIMS: Calcd for C40H72N3O26
D
(M+H+): m/z 1010.4398. Found: 1010.4390. Selected 13C NMR
D
(400 MHz, D2O, from HSQC) dH 103.2 (C-1_bGal), 102.6 (C-1_
a
Gal),
GalNAc), 50.8
(C-2_bGalNAc), 50.0 (CH2NH3þ), 49.2 (C-1_
aGalNAc).
101.9 (C-1_bGlc), 100.3 (C-1_bGalNAc), 93.4 (C-1_
a
Compound 10 (22 mg, 0.024 mmol) and 6 (40 mg, 0.028 mmol)
were dissolved in anhydrous CH2Cl2 (0.7 mL) containing molecular
sieves (50 mg). At ꢁ78 °C, N-iodosuccinimide (8 mg, 0.06 mmol)
and trifluoromethanesulfonic acid were added. The reaction was
warmed up to ꢁ30 °C for 1 h. Et3N was then added and the mixture
was purified by column chromatography on silica gel using 43%
EtOAc–hexane as eluent to give the compound 9 (45 mg, 88.6%
References
1. Forssman, J. Biochem. Z. 1911, 37, 78–115.
2. Hakomori, S.; Wang, S. M.; Young, W. W. PNAS 1977, 74, 3023–3027.
3. Ono, K.; Hattori, H.; Uemura, K. I.; Nakayama, J.; Ota, H.; Katsuyama, T. J.
Histochem. Cytochem. 1994, 42, 659–665.
4. Paulsen, H.; Bunsch, A. Angew. Chem., Int. Ed. Engl. 1980, 19, 902–903.
5. Nunomura, S.; Mori, M.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1989, 30, 6713–
6716.
6. Nilsson, U.; Ray, A. K.; Magnusson, G. Carbohydr. Res. 1994, 252, 137–148.
7. Zhang, J. B.; Kowal, P.; Fang, J. W.; Andreana, P.; Wang, P. G. Carbohydr. Res.
2002, 337, 969–976.
8. Houliston, R. S.; Bernatchez, S.; Karwaski, M. F.; Mandrell, R. E.; Jarrell, H. C.;
Wakarchuk, W. W.; Gilbert, M. Glycobiology 2009, 19, 153–159.
9. Shao, J.; Zhang, J. B.; Kowal, P.; Lu, Y. Q.; Wang, P. G. Biochem. Biophys. Res.
Commun. 2002, 295, 1–8.
yield). Rf: 0.35 (EtOAc/hexane 7:3). [a]
+125.0 (c 0.2, CHCl3). 1H
D
NMR (400 MHz, CDCl3) dH 7.52–7.14 (m, 45H), 5.80 (d, 1H,
J = 8.8 Hz), 5.44 (s, 1H), 5.36 (s, 1H), 5.31 (d, 1H, J = 2.0 Hz), 5.16
(d, 1H, J <1 Hz), 5.01 (d, 1H, J = 11.1 Hz), 4.97–4.81 (m, 5H), 4.81–
4.70 (m, 5H), 4.66–4.44 (m, 8H), 4.44–4.32 (m, 3H), 4.30–4.11
(m, 7H), 4.11–3.81 (m, 11H), 3.80–3.71 (m, 2H), 3.66–3.45 (m,
7H), 3.44–3.34 (m, 2H), 3.33–3.26 (m, 3H), 3.22 (t, 2H, J = 6.9 Hz),
2.58–2.51 (m, 1H), 2.16 (s, 3H), 2.03 (s, 3H), 1.92 (s, 3H), 1.72–
1.51 (m, 4H), 1.47–1.35 (m, 4H), 1.27 (s, 3H). 13C NMR (100 MHz,
CDCl3) dC 170.54, 170.44, 170.24, 170.21, 154.54, 139.40, 138.62,
138.37, 138.30, 138.16, 138.07, 138.05, 138.04, 137.37, 133.79,
129.41, 129.31, 129.28, 128.63, 128.48, 128.44, 128.42, 128.26,
128.00, 127.93, 127.90, 127.88, 127.71, 127.67, 127.62, 127.60,
127.56, 127.53, 127.46, 127.17, 126.39, 126.18, 103.62, 102.83,
101.34, 101.17, 100.50, 100.24, 95.38, 93.28, 82.32, 81.52, 81.20,
78.80, 76.92, 76.56, 76.38, 75.09, 74.86, 74.80, 74.62, 74.48,
73.45, 73.12, 72.84, 72.10, 69.75, 69.25, 68.97, 68.36, 68.33,
67.10, 66.85, 66.75, 65.44, 63.27, 61.20, 51.34, 29.59, 28.75,
26.50, 25.72, 22.35, 20.77, 20.70, 20.62. HRESIMS: Calcd for
10. Boysen, M.; Gemma, E.; Lahmann, M.; Oscarson, S. Chem. Commun. 2005, 3044–
3046.
11. Geng, Y. Q.; Zhang, L. H.; Ye, X. S. Chem. Commun. 2008, 597–599.
12. Zhang, P.; Ng, K.; Ling, C.-C. Org. Biomol. Chem. 2010, 8, 128–136.
13. Crich, D.; Li, W. J. J. Org. Chem. 2007, 72, 2387–2391.
14. Huang, L. J.; Wang, Z.; Li, X. N.; Ye, X. S.; Huang, X. F. Carbohydr. Res. 2006, 341,
1669–1679.
15. Yang, L.; Ye, X. S. Carbohydr. Res. 2010, 345, 1713–1721.
16. Manabe, S.; Ishii, K.; Ito, Y. J. Am. Chem. Soc. 2006, 128, 10666–10667.
17. Crich, D.; Vinod, A. U. J. Org. Chem. 2005, 70, 1291–1296.
18. Satoh, H.; Hutter, J.; Luthi, H. P.; Manabe, S.; Ishii, K.; Ito, Y. Eur. J. Org. Chem.
2009, 1127–1131.
19. Feng, J. H.; Ling, C.-C. Carbohydr. Res. 2010, 345, 2450–2457.
20. Andersen, S. M.; Ling, C.-C.; Zhang, P.; Townson, K.; Willison, H. J.; Bundle, D. R.
Org. Biomol. Chem. 2004, 2, 1199–1212.
21. Tanaka, N.; Ogawa, I.; Yoshigase, S.; Nokami, J. Carbohydr. Res. 2008, 343, 2675–
2679.
C
110H124N5O30Cl3Na (M+Na+): m/z 2122.7289. Found: 2122.7337.
22. Ellervik, U.; Magnusson, G. Tetrahedron Lett. 1997, 38, 1627–1628.
23. Nyffeler, P. T.; Liang, C. H.; Koeller, K. M.; Wong, C. H. J. Am. Chem. Soc. 2002,
124, 10773–10778.
24. Yan, R. B.; Yang, F.; Wu, Y. F.; Zhang, L. H.; Ye, X. S. Tetrahedron Lett. 2005, 46,
8993–8995.
4.29. 6-Aminohexyl 2-acetamido-2-deoxy-
syl-(1?3)-2-acetamido-2-deoxy-b- -galactopyranosyl-(1?3)-
-galactopyranosyl-(1?4)-b- -galactopyranosyl-(1?4)-b-
glucopyranoside, acetic acid salt (2)
a-D-galactopyrano-
D
a-
D
D
D-
25. Koeller, K. M.; Smith, M. E. B.; Wong, C.-H. Bioorg. Med. Chem. 2000, 8, 1017–
1026.
26. Kalikanda, J.; Li, Z. J. Org. Chem. 2011, 76, 5207–5218.
27. Ochiai, H.; Huang, W.; Wang, L. X. J. Am. Chem. Soc. 2008, 130, 13790–13803.
28. Lichtenthaler, F. W.; Kaji, E.; Weprek, S. J. Org. Chem. 1985, 19, 3505–3515.
Compound 9 (35 mg, 0.017 mmol) was dissolved in AcOH
(1.8 mL) and H2O (200 L) was added. The reaction was stirred at
90 °C for 2.5 h. The solution was then concentrated and the residue
was dissolved in anhydrous MeOH (4 mL) and NaOMe (100 L,
l
l