A highly convergent synthesis of an N-linked glycopeptide presenting the H-type 2 human blood group determinant

Article abstract of DOI:10.1016/j.tet.2006.02.080

The total synthesis of an H-type blood group determinant in a model biological setting is described. The construct is comprised of a high mannose core structure with projecting lactose spacers, culminating in a two-copy presentation of the H-type blood group determinant itself. Key reactions that were used in this construction include sulfonamidohydroxylation (see 15→18) and benzoate-directed glycosylation via an activated thiophenyl donor (see 34→36). Another key strategic element involved the epimerization of an interior core glucoside to reach the β-mannoside (see 37→38) required in the ring C sugar of the high mannose core.

Full text of DOI:10.1016/j.tet.2006.02.080

Tetrahedron 62 (2006) 4954–4978
A highly convergent synthesis of an N-linked glycopeptide
presenting the H-type 2 human blood group determinant
Zhi-Guang Wang,a J. David Warren,a Vadim Y. Dudkin,a Xufang Zhang,a
Ulrich Iserloh,a Michael Visser,a Matthias Eckhardt,a Peter H. Seebergera
and Samuel J. Danishefskya,b,
*
aLaboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue,
New York, NY 10021, USA
bDepartment of Chemistry, Columbia University, Havemeyer Hall, 3000 Broadway, New York, NY 10027, USA
Received 6 December 2005; revised 23 February 2006; accepted 27 February 2006
Available online 30 March 2006
We dedicate this paper to the magnificent accomplishments of Raymond Lemieux
Abstract—The total synthesis of an H-type blood group determinant in a model biological setting is described. The construct is comprised of
a high mannose core structure with projecting lactose spacers, culminating in a two-copy presentation of the H-type blood group determinant
itself. Key reactions that were used in this construction include sulfonamidohydroxylation (see 15/18) and benzoate-directed glycosylation
via an activated thiophenyl donor (see 34/36). Another key strategic element involved the epimerization of an interior core glucoside to
reach the b-mannoside (see 37/38) required in the ring C sugar of the high mannose core.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
envisioned that chemical synthesis could yet prove to be
the most viable way to provide the field of glycobiology
with ‘precision tools’ for the study of glycoproteins.6,7
Among the great variety of functions assumed by carbohy-
drates in biological systems, the role of the carbohydrate do-
main of glycoproteins in molecular recognition has been
a focus of particular attention for many scientific disci-
plines.1 Indeed, the growing field of glycobiology is devoted
to defining the effects of protein glycosylation on a range of
phenomena, including fertilization,2 tumor metastasis,3 and
immune response.4 Specific glycosylation patterns also
serve as markers for tumorigenesis and are used in blood pro-
filing.5 A major challenge facing the field of glycobiology is
the limited natural accessibility of homogeneous glycopep-
tides, which can be attributed to difficulties in isolation, com-
pounded by their often microheterogeneic nature. Certainly,
the development of technologies to allow access to homoge-
neous glycopeptides would be of significant value in probing
critical interactions at the interface of glycochemistry and
glycobiology. Despite the daunting challenges associated
with the preparation of glycopeptides in the laboratory, we
An ongoing focus of our laboratory has been the develop-
ment of methodologies that enable the preparation of com-
plex, fully synthetic glycopeptides.8 An early goal of this
research program was the assembly of pentacyclic glycopep-
tide, 1, in which the carbohydrate domain is N-linked to the
peptide through an asparagine g-carboxamide, not dissimi-
lar to the way Nature combines such structures. We describe,
herein, the design and execution of the synthesis of 1.9 We
note that, for reasons discussed below, we also accomplished
the synthesis of 2, which differs from 1 only in the absolute
stereochemistry of the peptide domain.10
As a further demonstration of the advances in the field of
glycopeptide synthesis, we set for ourselves the rather ambi-
tious ultimate goal of synthesizing an N-linked, 15-ring gly-
can construct (3), containing a mature H-type 2 blood group
determinant at its non-reducing end. Nothing of this scope
had previously been undertaken in synthesizing N-linked
glycopeptides. The manner in which we accomplished the
synthesis of this complex target compound is described
herein.11
* Corresponding author at present address: Laboratory for Bioorganic
Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York
Avenue, New York, NY 10021, USA. Tel.: +1 212 639 5502; fax: +1 212
772 8691; e-mail: s-danishefsky@ski.mskcc.org
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.02.080

Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
4955
OH
O
OH
HO
HO
O
HO
HO
HO
HO
OH
O
OH
O
O
O
HO
HO
HO
HO
O
O
HO
O
HO
O
O
O
O
O
O
O
HO
HO
NH
NH
HO
HO
AcN
H
AcN
H
HO
HO
HO
HO
AcN
H
AcN
H
HO
OH
NH2
OH
NH2
O
OH
O
OH
O
O
HO
O
O
O
O
O
O
H
N
H
H
N
H
N
N
N
N
N
H
N
N
H
N
H
H
H
H
H
H
O
CH3
O
O
O
CH3
O
O
1
2
HO
OH
O
HO
OH
O
HO
O
O
HO
O
HO
O
O
HO
O
O
HO
AcN
H
OH
AcN
H
HO
O
OH
O
OH
HO
HO
HO
HO
HO
OH
O
O
HO
HO
O
HO
O
O
O
O
HO
NH
HO
HO
AcN
H
AcN
H
HO
OH
O
OH
HO
O
OH
O
O
O
O
H
N
H
AcHN
O
N
NH2
AcHN
O
HO
O
O
HO
O
N
N
N
O
HO
H
H
O
H
H
O
O
CH3
O
O
O
OH
HO
HO
HO
OH
HO
3
2. Results and discussion
an efficient global deprotection sequence is critical to the
success of the enterprise.
In charting a program for glycopeptide synthesis, provisions
must be made for assembling the glycan domain. To the
extent that the ultimate goal is that of simulating the motifs
of Nature, the glycan synthesis itself may well be quite com-
plex. In addition, a polypeptide domain must be assembled.
Depending on the situation, the polypeptide domain may be
introduced as a single block, or in the form of segments to be
subsequently ligated. In either case, the need for the glycan
to be coupled to an appropriate peptide chain in the final
stages of the synthesis requires careful selection of appro-
priate glycosylation and protection strategies, with due
planning from the outset. Indeed, the implementation of
In the case of N-linked glycopeptides, the anomeric amine
required for eventual merger with a peptide-based acyl
donor can be generated in several forms (Fig. 1). Coupling
of the ‘per protected’ glycosylamine produces a glycopep-
tide, which is then subjected to global deprotection. While
the coupling step can be high yielding, maintenance of
anomeric configurational homogeneity of the N-linked
peptide may be problematic.12 Indeed, coupling of the
anomeric amino group of a perbenzylated glycan (path a)
or peracetylated glycan (path b) to the g-carboxyl of an
aspartyl residue on the peptide chain can produce a difficult
BnO
BnO
O
O
O
O
1) PeptideCOOH α, β
2) debenzylation
NH2
BnO
BnO
path a
AcN
H
AcN
H
NH2
HO
AcO
O
O
O
H
O
1) PeptideCOOH
2) deacylation
β
N
X
NH2
HO
AcO
path b
AcN
H
AcN
H
O
X = peptide or CH3
HO
O
O
1) PeptideCOOH
β
NH2
path c
HO
AcN
H
Figure 1. Comparison of glycopeptide assembly: (a) O-benzylated (electron-donating) glycosylamine, was coupled with peptide acid, followed by debenzy-
lation. In a complex glycosylamine system, peptide conjugation led to a/b mixture of glycopeptide due to anomerization of benzylated glycosylamine; (b)
O-acetylated glycosylamine (electron-deficient) was acylated with peptide acid. In this system, potential migration of acetyl from O/N led to the formation
of glycosyl acetamide (X¼CH3) in addition to the desired b-glycopeptide; (c) selective N-acylation of a polyhydroxyl glycosylamine can be achieved with
peptide acid utilizing minimum protection.

4956
Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
to separate mixture of N-aspartyl anomers or anomeric
acetamide.
through various glycal-based methods.14,15 Following global
deprotection, the glycan would be aminated at its reducing
end, using the procedure developed by Kochetkov,16 and
then subjected to conjugation with the peptide (vide infra)
as reported by Lansbury.13
In this connection, we note that in an earlier initiative in our
laboratory, we had accomplished the maximally convergent
synthesis of an N-linked glycopeptide.12 A pentacyclic gly-
can was prepared using methodology instituted under our
paradigm of glycal assembly. Even in this complex setting,
the glycal linkage was convertible to the required terminal
glucosamine linkage. In the final stage of that effort, reduc-
tion of b-anomeric azide 4, in the context of a substantially
protected glycan, produced the glycosylamine. Following
aspartylation with tripeptide and pentapeptide donors and
then global deprotection, high mannose tripeptide 5 and
pentapeptide 6 were each isolated as 2:1 (b:a) anomeric
mixtures. The precise point at which anomeric configura-
tional integrity was compromised (cf. inter alia, reduction
of the azide, acylation or deprotection) is not clear.
As in our previously reported syntheses of 5 and 6,12 we
hoped that a terminal glycal (see structure type 10) would
prove suitable for the eventual presentation of the 1-b-amino
function of the A ring (N-acetylglucosamine) to the peptide,
as required under path c (Fig. 1). The pentasaccharide glycal
would be obtained with high convergence by coupling of
a donor derived from ‘pre-trimannose glycal’ 7 with pre-
chitobiose glycal 8. Both 7 and 8 can be accessed in short
order from D-glucal.12,15
However, a significant issue presents itself in this otherwise
attractive coupling strategy. The glycal assembly method,
CO2H
H
HO
NH
OH
O
OH
O
BnO
HO
O
BnO
HO
BnO
HO
OH
O
OH
O
HN
O
O
O
steps
BnO
HO
PMBO
HO
O
O
BnO
O
HO
O
O
O
O
O
O
O
H
N
BnO
HO
N3
BnO
HO
BnO
BnO
BnO
AcN
H
HO
AcN
H
*
AcN
H
AcN
H
NHR
HO
O
OH
O
OH
O
HO
4
5: R = H
* 2:1 (β:α)
6: R = LeuAla
Fortunately, as demonstrated by Lansbury13 fully depro-
tected glycans bearing b-configured anomeric amino func-
tions can be efficiently acylated, taking advantage of the
significantly higher nucleophilicity of the amino group
(Fig. 1, path c). The stereochemical integrity of the reducing
terminus is preserved in the case of fully deprotected gly-
cans. Furthermore, use of fully deprotected free glycans
and glycosylamines obviates the need for late stage global
deprotection after formation of the glycopeptide.
which we favored, was well suited to fashion the C-ring as
a b-glucoside (cf. 9) rather than as the requisite b-mannoside.
The high convergence of our proposed synthesis seemed suf-
ficiently attractive to justify, if necessary, an indirect route to
reach the b-mannoside BC connection. The thought was to
exploit the expected ease of first appending the C-ring as
a b-glucoside. This would be followed by epimerization of
the C2 center of the C-ring (see asterisk, structure 9), thereby
producing the mannoside (10). In essence, the question was
whether the efficiency of the assembly phase, enabled by gly-
cal logic, would vindicate the awkwardness of the need for
a late stage gluco/manno epimerization sequence (Fig. 2).
In planning the synthesis of the five-ring glycopeptide 1, we
anticipated that the required glycan might be assembled
OP2
PO
OP2
O
PO
O
PO
PO
PO
PO
PO
HO
O
O
O
PO
D
O
C
O
O
O
PO
O
O
+
O
PO
PO
B
PO
O
PO
PO
O
P3N
H
O
O
PO
PO
PO
PO
*
OH
PO
D
P3N
H
PO
7
8
PO
PO
OP2
9
A
O
OP2
OP2
OH
O
PO
PO
HO
HO
O
PO
O
PO
HO
OP1
O
OH
O
O
O
O
PO
HO
O
PO
HO
O
HO
O
O
O
O
O
O
O
PO
HO
R
PO
P3N
H
HO
PO
HO
AcN
H
AcN
H
PO
PO
HO
HO
O
OH
10
OP2
11: R = β-NH2
12: R = α/β-NH2
Figure 2. Synthetic plan for the core pentasaccharide glycosylamine. P, P1, P2, and P3 represent protecting groups.

Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
4957
PMP
O
HO
PMBO
HO
HO
a
2.1. Discussion of results
b
O
O
O
O
HO
HO
HO
Our venture commenced with the preparation of the building
blocks from which the core pentasaccharide glycan (cf. 11)
could be synthesized. From the outset, we recognized that it
would be important to develop a program that would allow
for maximum diversification during the assembly of the ma-
ture glycan. Hopefully the strategy would not only encom-
pass this endeavor, but would also establish a framework
for future projects. As shown in Schemes 1 and 2, D-glucal
(13) would serve as the common starting material for each
building block. Additionally, the synthesis would make use
of a tactic for azaglycosylation that had been developed in
our laboratory some 15 years ago.17
20
21
13
OTBS
O
BnO
BnO
BnO
BnO
BnO
BnO
BnO
d
c
O
O
BnO
SEt
SEt
BnO
OH
22
23
24
OMe
OR
BnO
BnO
BnO
O
BnO
O
e
O
O
BnO
BnO
SEt
25
26 R = H
27 R = TBS
28 R = MBz
f
g
Scheme 2. Synthesis of the a-mannoside donor and acceptor from D-glucal:
(a) p-anisaldehyde dimethyl acetal, PPTS, THF, 48%; (b) Dibal-H, DCM,
75%; (c) 1. DMDO, DCM, 2. EtSH, TFAA, 89% in two steps; (d) 1.
DMSO, Ac2O, 2. NaBH4, DCM, MeOH, 76% in two steps, 3. TBSOTf,
DCM, Et3N, 93%; (e) 1. EtSH, HgBr2, CH3CN, 95%, 2. NaOMe, MeOH;
(f) TBSOTf, DCM, Et3N, 93%; (g) p-toluoyl chloride, DCM, Et3N, 75%.
The synthesis of the pre-chitobiose glycal (cf. 19) com-
menced with 3,6-dibenzylglucal 14, which had in turn
been synthesized from D-glucal (13) as previously reported
(Scheme 1).17 Acetylation of the free alcohol in 14 provided
the fully protected glucal 15. Iodosulfonamidation followed
by thiolysis of the intermediary aziridine at the anomeric
center gave rise to thiodonor 17. The original glucal 14
then served as an acceptor in the methyl triflate promoted
glycosylation with 17. Following methanolysis of the
C40 acetate, the pre-chitobiose glycal acceptor 19 was in
hand.
Alternatively, monosaccharides of this type may be prepared
from the commercially available orthoester 25 in three steps,
beginning with mercuric bromide-mediated ring opening
with ethanethiol followed by methanolysis of the resulting
acetate to afford 26. The free alcohol of 26 can be repro-
tected with TBS triflate or p-toluoyl chloride (4-methylben-
zoyl chloride) to afford 27 or 28, respectively. It should be
noted that this alternate route is more attractive due to its
amenability to the production of large quantities of the
a-mannoside donor.
Our most convergent route to the requisite pre-trimannose
glycal of the type 7 would be through the selective introduc-
tion of a-mannosyl residues at C3 and C6 of glycal 21. The
synthesis of 21 was accomplished in a two-step sequence
starting, once again, from D-glucal (13). Thus, engagement
of the C4 and C6 alcohols of 13 as a p-methoxybenzylidene
derivative gave rise to 20 (Scheme 2). Reductive cleavage
of the acetal linkage afforded the appropriately protected
glycal 21.
With the mono- and disaccharide building blocks in hand,
we began assembly of the pentasaccharide (cf. 11), and ulti-
mately construction of the target pentasaccharide–pentapep-
tides 1 and 2. This exercise would serve to field-test and,
indeed, validate our plan for synthesizing complex glyco-
polypeptides carrying biological information (cf. 3). Thus,
the construction of the a-trimannoside donor commenced
with di-a-mannosylation of the glycal diol acceptor 21
(Scheme 3). Diglycosylation with 2-O-TBS protected thio-
mannoside 27, using methyl triflate as the activating agent,
afforded trisaccharide glycal 30 in 54% yield. The same
transformation proceeded in somewhat higher yield (63%)
to afford 29 when ester-protected thiomannoside 28 was
used as the donor. However, because this ester group would
later interfere with the epimerization sequence required at
The required a-mannoside donor, 24, was synthesized from
commercially available tri-O-benzyl-D-glucal (22). Thus,
stereoselective epoxidation with dimethyldioxirane (DMDO),
followed by selective ring opening with ethanethiol, in the
presence of trifluoroacetic anhydride, gave rise to thiogly-
coside 23. Inversion of the alcohol at C2 was accomplished
by Moffat-like oxidation18 followed by stereoselective re-
duction of the resulting ketone with sodium borohydride.
The now inverted alcohol was then protected to afford
thiodonor 24.
I
HO
HO
HO
BnO
BnO
AcO
BnO
BnO
AcO
BnO
O
b
c
O
O
O
RO
SEt
BnO
PhSO2N
H
PhSO2N
H
14 R = H
15 R = Ac
16
17
13
a
BnO
AcO
BnO
BnO
O
BnO
O
O
BnO
d
e
HO
O
O
14 + 17
O
BnO
BnO
BnO
PhSO2N
H
PhSO2N
H
18
19
Scheme 1. Synthesis of the pre-chitobiose glycal acceptor building block from D-glucal: (a) Ac2O, pyridine, DMAP, 94%; (b) IDCP (Iodonium-di-sym-collidine
perchlorate), PhSO2NH2, DCM, 0 ꢀC, 94%; (c) EtSH, LHMDS, DMF, ꢁ40 ꢀC–0 ꢀC, 62%; (d) MeOTf, DTBP (2,6-di-tert-butylpyridine), DCM, 0 ꢀC to rt,
70%, b/a 6:1; (e) NaOMe, MeOH, 90%.

4958
Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
OR
BnO
OTBS
O
O
BnO
BnO
BnO
BnO
BnO
OR
O
HO
PMBO
HO
BnO
BnO
BnO
O
c
d
O
a
O
O
+
PMBO
O
PMBO
O
O
SEt
BnO
O
21
BnO
BnO
O
BnO
BnO
O
OR
BnO
OTBS
27 R = TBS or
28 R = MBz
29: R = MBz
30: R = TBS
b
31
OTBS
O
BnO
OTBS
O
BnO
HO
BnO
O
BnO
O
BnO
BnO
O
BnO
BnO
BnO
BnO
PhSO2N
H
i
O
O
O
BnO
19
PMBO
O
O
BnO
PMBO
O
O
O
SEt
O
O
BnO
OR
g
BnO
OR
*
PhSO2N
H
BnO
BnO
BnO
O
BnO
O
OTBS
BnO
BnO
OTBS
35: R = Ac
36: R = Bz
37: R = H
32: R = H
33: R = Ac
34: R = Bz
e
h
f
h
OTBS
O
OTBS
BnO
BnO
BnO
O
BnO
BnO
BnO
OH
O
OAc
O
j
O
O
BnO
O
BnO
PMBO
PMBO
O
BnO
O
O
BnO
O
O
O
O
O
O
BnO
BnO
BnO
BnO
BnO
BnO
PhSO2N
H
BnO
PhSO2N
BnO
O
O
Ac
BnO
BnO
OTBS
OTBS
38
39
Scheme 3. Synthesis of the core pentasaccharide glycal: (a) 27 or 28, MeOTf, DTBP, DCM, 54% for 30, 63% for 29; (b) 1. NaOMe, MeOH, 77%, 2. TBSOTf,
Et3N, DCM, 95%; (c) DMDO, DCM, crude yield >99%; (d) EtSH, TFAA, DCM, 76%; (e) Ac2O, Et3N, DCM, 91%; (f) BzCl, pyridine, DCM, 90%; (g) 19,
MeOTf, DTBP, DCM, 64% for 35, 71% for 36; (h) LiAlH4, Et2O; (i) 1. Dess–Martin periodinane, DCM, 2. L-Selectride, THF; (j) Ac2O, pyridine, DMAP, DCM,
82% in four steps.
the pentasaccharide stage, the ester protecting groups in 29
had to be converted to silyl groups (29/30). While this
two-step sequence could easily be achieved, the overall route
was not highly efficient.
overall epimerization at C2 (compound 37, see asterisk).20
The overall inversion at this center would require gaining ac-
cess to a C-ring uloside (i.e., 2-keto) functionality which
could subsequently be reduced to provide, hopefully, a C2
axial alcohol (cf. 38). In the event, deprotection of neither
the acetate in 35 or the benzoate in 36 could be achieved un-
In either case, the double bond of the trisaccharide 30 was ep-
oxidized with DMDO to afford 31. Unfortunately, the direct
coupling between this epoxide as a donor, and the pre-chito-
biose acceptor 19 was never successful. Despite significant
efforts to optimize this type of transformation, only trace
amounts of pentasaccharide product could be detected by
this seemingly straightforward approach. Fortunately we
had earlier developed a modality to deal with such a contin-
gency.9,19 Applied to the case at hand, epoxide 31 was trans-
formed into the thioglycoside alcohol 32, which was
subsequently protected as either an acetate (33) or benzoate
(34). Both the b-glucoside bond donors were evaluated in
coupling with the acceptor 19. Coupling with trisaccharide
acetate 33 provided corresponding pentasaccharide 35, albeit
in modest yield (64%). In practice, glycosylation was signif-
icantly complicated by orthoester formation. By contrast,
upon using benzoate thioglucoside 34 as donor, the reaction
was quite reproducible, providing pentasaccharide 36 in
a more acceptable 71% yield. Not surprisingly, the benzoate
proved to be a superior trans-glycosylation directing group.
der Zemplen conditions.21 However, either acyl group could
´
easily be cleaved reductively with LAH, yielding alcohol 37.
The equatorial alcohol in 37 was then oxidized with Dess–
Martin periodinane,22 giving rise to an unstable ketone that
was immediately reduced using L-Selectride.23 In this way,
alcohol 38, with the C2-axial hydroxyl group, was obtained
in 82% yield over four steps. Subsequent acylation of the
alcohol afforded diacetate 39.
At this point, appropriate functionalization of the reducing
end glycal and global deprotection had to be achieved prior
to introduction of the peptide chain. Toward this end, glucal
39 was subjected to iodosulfonamidation and thiolysis,
thereby giving rise to phenyl thioglycoside 40 in 85% yield
(Scheme 4). The latter was subjected to TBAF-mediated de-
silylation, and the resultant crude product, 42, was reacted
with sodium in liquid ammonia, followed by peracetylation
of the fully deprotected glycan. Unfortunately, as spectro-
scopic analysis of the product demonstrated, the erstwhile
thiophenyl group was no longer present, having presumably
been cleaved during the dissolving metal reduction. The
observed product of this sequence, 44, contained two
anomeric protons.
The proposal had indeed been realized in a highly conver-
gent fashion. However, it would now be necessary to convert
the C-ring from gluco to the manno stereochemistry, by

Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
4959
OTBS
O
OH
BnO
BnO
O
BnO
BnO
BnO
BnO
OAc
O
OAc
O
a
O
b
O
BnO
O
BnO
O
39
PMBO
PMBO
O
O
BnO
BnO
O
O
O
O
O
O
BnO
BnO
SR
SR
BnO
BnO
BnO
PhSO N
BnO
BnO
PhSO N
2
2
PhSO N
2
PhSO N
BnO
2
H
O
OTBS
O
OH
Ac
Ac
H
BnO
BnO
40: R = Ph
41: R = Et
42: R = Ph
43: R = Et
OAc
O
OH
O
AcO
HO
AcO
AcO
HO
HO
OAc
O
OH
O
c
O
AcO
O
d
O
AcO
HO
HO
O
O
AcO
HO
O
O
O
O
O
O
O
AcO
HO
R
SEt
AcO
HO
AcN
H
AcN
H
AcO
AcO
HO
AcN
H
AcN
H
HO
O
OAc
O
OH
AcO
HO
44: R = H
45: R = SEt
46
Scheme 4. Upgrading of the pentasaccharide terminal glycal: (a) 1. IDCP, PhSO2NH2, DCM, 2. PhSH, LHMDS, DMF, 85% in two steps for 40, or EtSH,
LHMDS, DMF, 81% in two steps for 41; (b) TBAF, THF, 80%; (c) 1. Na/NH3, THF, 2. Ac2O, pyridine, DMAP, 70% for 44, 77% for 45; (d) NaOMe,
MeOH, 86%.
We reasoned that the phenyl ring of the anomeric thiophenyl
function could well have facilitated the reduction of the C–S
linkage. Accordingly, it was surmised that an alkyl thiogly-
coside might be more stable under the same reductive condi-
tions. In the event, when ethylthioglycoside 43 (obtained
from glucal 39 in a similar sequence) was subjected to the
action of sodium in liquid ammonia followed by peracetyla-
tion and purification, the desired product 45, containing an
intact C–S bond, was isolated in 77% yield. The O-esters
in 45 were then saponified to provide the fully deprotected
pentasaccharide 46 in 86% yield.
on the evaluation of structural interactions within carbohy-
drate or peptide domains; however, little is known about
communication across domains.25 This gap can be attrib-
uted, in large part, to the very limited accessibility of homo-
geneous glycopeptide constructs. Given our interest in
assembling such constructs through convergent chemical
synthesis, we chose to prepare 1 and 2 in order to probe ques-
tions of stereochemical communication between the carbo-
hydrate and peptide domains.10
Indeed, the results of high-field NMR studies performed on
1 and 2 strongly indicate the existence of stereochemical
interactions between the peptide and carbohydrate domains
of these N-linked glycopeptides. Although the NOESY pat-
terns of compounds 1 and 2 were quite similar, as ex-
pected, distinct shift differences were observed for the
protons at the central amino acid residues, as indicated in
Figure 3. Thus, distinct shifts were observed for the amide
NH protons of asparagine (to which the carbohydrate is
linked) and the adjacent valine residue. Since amide proton
chemical shifts are good indicators of peptide backbone
conformations, these results clearly demonstrated that
a global change in peptide stereochemistry impacts the
structural conformation of the glycopeptide.26 Importantly,
the results of this exercise show that cross-domain com-
munication cannot be solely attributed to the bulk of the
carbohydrate moiety.10
In order to implement a Kochetkov–Lansbury sequence for
building the glycopeptide domain, it would be necessary to
liberate, in some as yet unspecified fashion, an anomeric hy-
droxyl group. Thus, overall hydrolysis of the thioglycoside
functionality in 46 would perhaps lead us to 11. Though
reaction of 46 with NBS in aqueous THF turned out to be
difficult to reproduce, hydrolysis with HgCl2 proceeded
efficiently24 to afford the reducing saccharide 47 in 95%
yield, after gel filtration. Finally, this alcohol was converted
into b-anomeric amine 11 using the Kochetkov protocol;16
1
no a-anomer was detected in the H NMR spectrum of the
product.
Two pentapeptide enantiomers, one composed entirely of
L-amino acids (48) and the other of D-amino acids (49),
were prepared using an automatic Fmoc-synthesizer on a
Rink amide MBHA resin. Each of the peptides was conju-
gated with the amine 11 using HOBt/HBTU to activate the
aspartic acid. The diastereomeric b-N-linked glycopeptides
1 and 2 were isolated in 40% yield, with no observable for-
mation of the a-anomer (Scheme 5).
Having field-tested the technologies required for glycal as-
sembly and peptide conjugation in the syntheses of glyco-
peptides 1 and 2, we were prepared to take on the
challenge of synthesizing our ultimate target compound:
a pentadecasaccharide glycan construct linked to a mature
H-type 2 blood determinant (3). As will be seen, many
new difficulties had to be overcome to meet this challenge.
We turn first to our approach to the synthesis of the glycal
portion of the molecule (50). In this system, the H-type 2 tri-
saccharides (shown in green) are appended to the common
pentasaccharide domain (shown in blue) through two lactos-
amine spacer units (shown in red).
At this point, it is appropriate to discuss our reasons for pre-
paring not only glycopeptide 1, but also glycopeptide 2,
which differs only in the absolute stereochemistry of the
peptide domain. An important area of study in the field of
glycobiology is the evaluation of the structural configura-
tions of glycopeptides. Early work in this field has focused

4960
Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
OH
O
OH
HO
HO
O
HO
HO
HO
HO
OH
O
OH
O
O
O
HO
HO
a
HO
HO
O
O
46
HO
O
b
HO
O
O
O
O
O
O
O
HO
HO
OH
NH2
HO
HO
AcN
H
AcN
H
HO
HO
HO
HO
AcN
H
AcN
H
HO
O
O
HO
OH
OH
47
11
OH
HO
OH
O
HO
HO
OH
NH2
O
O
O
O
OH
O
O
H
N
H
HO
c
11
+
HO
N
O
HO
O
O
H
N
O
N
H
N
H
N
O
HO
O
H
H
HO
O
CH3
O
O
HO
AcN
AcN
HO
H
O
OH
H
HO
48
7.6 Hz
H
CH3
H
H
5.5 Hz
N
1
HN
O
O
O
O
HN
H
8.0 Hz
8.5 Hz
H
5.9 Hz
NH
O
HO
O
NH
H
H2N
OH
O
HO
OH
HO
HO
OH
NH2
O
OH
O
O
O
O
O
H
N
H
c
HO
O
HO
O
N
HO
11
+
O
H
N
O
N
H
N
N
H
O
HO
O
H
H
HO
HO
AcN
H
O
CH3
O
O
AcN
H
HO
O
OH
HO
49
7.0 Hz
H
CH3
H
H
5.7 Hz
N
2
HN
O
O
O
H
NH
HO
HN
H
O
8.1 Hz
8.3 Hz
6.4 Hz
O
NH
H
O
H2N
Scheme 5. Construction of glycopeptides: (a) HgCl2, CaCO3, H2O, 95%; (b) NH4HCO3, H2O, >99%; (c) 48 or 49, HOBt, HBTU, iPr2NEt, DMSO, 40%.
HO
HO
OH
O
HO
O
OH
O
HO
O
O
HO
O
O
HO
O
O
HO
AcN
H
OH
AcN
H
HO
O
OH
OH
O
BnO
O
OH
HO
BnO
HO
HO
HO
HO
BnO
OH
O
OAc
O
O
O
HO
O
BnO
O
HO
PMBO
O
HO
O
BnO
O
O
O
O
O
O
HO
BnO
HO
BnO
AcN
H
HO
BnO
PhSHO2N
H
HO
BnO
OH
O
O
OH
HO
BnO
OH
O
50
51
AcHN
AcHN
O
HO
O
O
HO
O
O
HO
O
O
O
O
OH
HO
HO
HO
OH
HO
A logical disconnection, shown in structure 50 (see lines),
would lead us to the common pentasaccharide domain as
a key building block. The appropriately protected pentasac-
charide intermediate (51) would be easily obtained through
desilylation of the previously synthesized 39 (Scheme 3).
At this stage, two approaches for the preparation of the
pentadecasaccharide 50 were considered. In the first, the di-
and trisaccharide precursors 52 and 53 would be assembled
to form the H-type 2-lactosamine donor pentasaccharide
54. The latter would then be joined with 51 through

Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
4961
The more convergent ‘5+5+5’ approach was investigated
first. Unfortunately, the projected 5+5+5 coupling reaction
did not occur cleanly upon activation of either the thio-
ethyl-, fluoro- or trichloroacetimidate derivatives of donor
54. However, on the basis of mass spectral analysis, it
appeared that 5+5 coupling did in fact occur at each of the
acceptor sites in 51. The stereochemistry of these gly-
cosylations could not be determined in the context of the
inhomogeneous product. However, in no fraction could we
claim mass spectral evidence that the 15-mer had arisen
from an actual 5+5+5 coupling.
2
Since at least one glycosylation had occurred, we surmised
that each of the glycosylation acceptor sites in 51 was,
in principle, a competent acceptor. Perhaps with a more
reactive glycosyl donor, two-fold glycosylation could be
achieved. We reasoned that smaller, properly activated lac-
tosamine donors, such as 56, might allow for two-fold glyco-
sylation of 51. With proper selection of the lactosamine
protecting groups, two new acceptor sites could be unveiled
subsequent to coupling with 51, at which point two activated
H-type 2 donors, such as 55, could be concurrently installed,
producing the pentadecasaccharide via a ‘5+2+3’ coupling
strategy (Scheme 6b).
1
8.8
8.7
8.6
8.5
8.4
8.3
8.2
8.1
ppm
Figure 3. Amide proton spectra of 1 and 2 recorded at 800 MHz at 5 ꢀC.
Samples were dissolved in 90% H2O/10% D2O ([1]¼5 mM, [2]¼1 mM)
and the pH was adjusted with a 10 mM phosphate buffer (1: pH 3.5, 2:
pH 4.2). The H2O signal was suppressed using the Watergate method.
With these considerations in mind, we chose compound 60
as our lactosamine donor. This compound incorporates an
orthogonally protected hydroxyl group at C30 as well as
a strongly b-directing phthalamide at C2. The synthesis of
60 commenced with the hexaacetyllactal 57 (Scheme 7).
This compound was subjected to deacetylation with am-
monia in methanol, and the resulting hexaol was selectively
allylated at C30 through the intermediacy of dibutyltin acetal
formation.27 Benzylation of the remaining hydroxyl groups
afforded lactal 58 in 50% yield over four steps. The
reducing end was then functionalized using the previously
developed silylethylsulfonamide (SES) methodology.28
diglycosylation, as shown, in a highly convergent ‘5+5+5’
coupling reaction (Scheme 6a). A second, more linear, ap-
proach would involve sequential coupling of the di- and tri-
saccharide precursors to the initial pentasaccharide unit (51).
Thus, an appropriately protected lactosamine derivative 56
would be coupled to 51. Then, following selective deprotec-
tion, diglycosylation with the H-type 2 trisaccharide would
occur to afford the glycal 50 (Scheme 6b).
a) 5+5+5 strategy
O
OBn
O
O
OH
BnO
I
O
BnO
BnO
O
O
O
BnO
BnO
BnO
OBn
O
BnO
O
BnO
OBn
O
O
OAc
O
BnO
O
O
BnO
O
O
BnO
O
PhSO2N
H
O
PMBO
O
BnO
O
BnO
O
O
X
+
OBn
O
BnO
O
50
BnO
PhSO2N
H
BnO
52
BnO
PhthN
OBn
BnO
BnO
BnO
BnO
PhSO2N
H
O
OBn
O
OH
54: X = SEt, F, trichloroimidate
+
51
OBn
BnO
BnO
HO
OBn
O
BnO
O
O
BnO
BnO
53
b) 5+2+3 strategy
OH
O
BnO
BnO
BnO
BnO
BnO
OBn
O
BnO
HO
OBn
O
OAc
O
BnO
O
O
O
BnO
BnO
PMBO
O
O
BnO
O
SEt
O
O
BnO
O
O
SEt
50
O
BnO
BnO
PhthN
BnO
BnO
PhthN
BnO
PhSO2N
H
O
BnO
O
OH
OBn
BnO
55
56
51
OBn
BnO
Scheme 6. Assembly of divalent H-type 2 antigen-encoded 15-mer glycal: comparison of the 5+5+5 and 5+2+3 strategies.

4962
Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
Thus, iodosulfonamidation with 2-trimethylsilylethylsulfo-
namide, followed by thiolysis, with concurrent migration
of sulfonamide from C-1 to C-2 and ejection of iodide, pro-
vided thioglycoside 59 in 85% yield (two steps). Finally,
the sulfonyl and allyl protecting groups were converted to
phthaloyl29 and chloroacetyl, respectively, to provide the
desired lactosamine donor 60.
zation of the reducing end, we field-tested the deprotection
sequence on the unfunctionalized glycal 67. Thus, the
phthaloyl and O-acetate groups were first removed using
ethylenediamine. Next, the sulfonamide, p-methoxybenzyl,
and benzyl linkages were reductively cleaved with sodium
in liquid ammonia. The crude product was peracetylated,
purified through normal phase chromatography, and then
BnO
AllO
BnO
MCAO
OBn
O
OBn
O
BnO
AllO
AcO
AcO
OBn
O
OAc
O
BnO
O
BnO
O
O
BnO
O
O
AcO
a
b
c
O
O
SEt
SEt
O
BnO
BnO
BnO
BnO
BnO
AcO
NH
BnO
PhthN
AcO
SO2
TMS
59
60
57
58
Scheme 7. Synthesis of the lactosamine building block: (a) 1. NH3, MeOH, 2. Bu2SnO, PhH, 3. allyl bromide, Bu4NBr, 4. BnBr, NaH, DMF, 50% in four steps;
(b) 1. IDCP, TMSCH2CH2SO2NH2, 75%, 2. EtSH, LiHMDS, DMF, 85%; (c) 1. CsF, DMF, 90 ꢀC, 94%, 2. phthalic anhydride, pyridine, then Piv2O or Ac2O,
>99%, 3. RhCl(PPh3)2, DABCO, THF; 1 N HCl, 74%; 4. (ClCH2CO)2O, DTBP, cat. DMAP, DCM, 97%.
´
deacetylated under Zemplen conditions, to afford fully de-
protected glycal 50.
Glycal assembly was once again used to prepare the H-type
2 trisaccharide donor 55 (Scheme 8). Galactal 61 was selec-
tively a-epoxidized with DMDO and the resulting epoxide
was directly coupled with 3,6-dibenzylglucal 14 in the pres-
ence of ZnCl2. The newly formed C20 hydroxyl in the result-
ing disaccharide was fucosylated with 62, to furnish
trisaccharide glucal 63 in 50% yield (three steps). Perbenzy-
lation and functionalization of the reducing end, following
the procedure described for lactosamine 60 (vide supra),
provided the 2-N-phthaloyl thioglycoside donor 55 required
for the introduction of H-type 2 specificity.
Having established the feasibility of the deprotection se-
quence with the glycal, 67, we sought to apply the same
logic to the deprotection of the functionalized glycan.
Thus, iodosulfonamidation of glycal 67 gave the trans–
diaxial adduct 68, which was then subjected to thiolysis
with LiSEt in the hope of obtaining the corresponding thio-
glycoside 69 (Scheme 10). However, this transformation
failed to provide 69; instead, the reducing hemiacetal 70
O
OTIPS
O
O
BnO
HO
O
BnO
O
BnO
BnO
OBn
O
OTIPS
OBn
O
O
BnO
O
BnO
O
O
BnO
BnO
O
O
O
O
14
a
b
c
BnO
O
O
61
SEt
SEt
"OH"
BnO
BnO
BnO
O
O
O
PhthN
NH
F
SO2
O
O
O
O
OBn
55
OBn
63
OBn
64
OBn
OBn
OBn
BnO
OBn
BnO
TMS
OBn
BnO
BnO
62
Scheme 8. Synthesis of the wing trisaccharide 55: (a) 1. 61, DMDO, DCM; 2. 14, ZnCl2, DCM; 3. 62, SnCl2, AgOTf, DTBP, 50% (three steps); (b) 1. TBAF,
THF, 2 h then K2CO3, MeOH; 2. BnBr, NaH, DMF, 85% (two steps); 3. IDCP, TMSCH2CH2SO2NH2; 4. EtSH, LiHMDS, DMF ꢁ40 ꢀC to 0 ꢀC, 75% (two
steps); (c) 1. CsF, DMF, 100 ꢀC, 5 d, 65%; 2. phthalic anhydride, pyridine; 3. Ac2O, 97% (two steps).
With the required subunits and technology in hand, we
turned to the assembly of the pentadecasaccharide glycal.
Thus, the C2 hydroxyl groups in 39, previously protected
with silyl groups, were now unveiled with TBAF to afford
diol 51. The latter was coupled with excess lactosamine do-
nor 60, using methyl triflate as a promoter (Scheme 9).30 Im-
portantly, the diglycosylation of 51, which had failed with
pentasaccharide donor 54, could be easily achieved with di-
saccharide donor 60, to provide the desired nonasaccharide
65 in 62% yield. Next, the chloroacetate groups at C30 of
the lactosamine units in 65 were removed under neutral con-
ditions, while keeping the acetate on the central mannose
unit and the terminal glycal intact. Finally, the H-type 2
trisaccharides (55) were introduced and, once again, di-
glycosylation proceeded smoothly to give rise to pentadeca-
saccharide 67 in 78% yield.
was isolated from the reaction mixture in 40% yield. In
fact, we were never able to gain access to 69, whereas
the hydrolysis product 70 was isolated even under strictly
anhydrous conditions.
As we were unable to convert the glycal into the reducing
end thioglycoside in the context of the pentadecasaccharide,
we decided to investigate the possibility of direct global
deprotection of the oligosaccharide hemiacetals under dis-
solving metal reduction conditions. Such conditions, while
clearly too harsh for the hemiacetal in the open aldehyde
form, may leave the ‘reducing end’ intact provided that it
is ‘protected’ via total dominance of the ring closed form
in liquid ammonia. The results of our model studies (summa-
rized in Table 1) demonstrate the truly remarkable stability
and survivability of the reducing oligosaccharides under
these conditions. A series of reducing glycans, including ex-
amples with amides and hydroxyls at C-2, were deprotected
with yields of isolated peracetylated products typically
above 70%.31
At this stage, the challenge was that of achieving global
deprotection of the pentadecasaccharide system. Though
our ultimate strategy was to deprotect following functionali-

Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
4963
BnO
RO
OBn
O
BnO
O
O
O
BnO
BnO
PhthN
BnO
OR
O
BnO
BnO
MCAO
OBn
O
BnO
BnO
O
BnO
O
BnO
O
BnO
OAc
O
SEt
BnO
O
BnO
BnO
O
OAc
O
PMBO
PhthN
O
O
BnO
O
O
BnO
O
60
PMBO
O
BnO
O
BnO
O
O
O
BnO
BnO
PhSO2N
R'
BnO
BnO
BnO
b
BnO
PhSO2N
H
O
OR
BnO
BnO
O
BnO
39 R = TBS, R' = Ac
51 R, R' = H
a
PhthN
65: R = MCA
66: R = H
BnO
BnO
O
c
O
RO
O
O
BnO
OBn
BnO
BnO
BnO
OBn
O
BnO
OBn
O
BnO
BnO
O
O
O
BnO
BnO
O
O
OBn
O
BnO
O
BnO
O
PhthN
BnO
BnO
PhthN
BnO
O
O
O
SEt
BnO
OBn
O
PhthN
O
OBn
BnO
BnO
BnO
BnO
BnO
O
OBn
OAc
O
O
OBn
BnO
BnO
55
PMBO
BnO
O
e
O
O
O
O
BnO
d
BnO
BnO
PhSO2N
H
BnO
OBn
O
BnO
OBn
O
PhthN
67
PhthN
O
BnO
O
O
BnO
O
O
BnO
O
O
O
O
OBn
BnO
BnO
BnO
OBn
BnO
HO
HO
OH
O
HO
OH
O
HO
O
HO
O
O
O
O
HO
O
O
HO
AcN
H
OH
AcN
O
H
OH
HO
HO
HO
O
OH
HO
HO
HO
OH
O
O
HO
HO
O
HO
O
O
O
O
HO
HO
HO
HO
HO
AcN
H
OH
O
OH
O
50
AcHN
O
AcHN
O
OH
O
O
O
HO
O
O
HO
O
O
HO
HO
OH
HO
OH
HO
Scheme 9. Synthesis of the 15-mer glycal: (a) TBAF, THF, 77%; (b) 60, MeOTf, DTBP, DCM, 0 ꢀC to rt, 62%; (c) thiourea, NaHCO3, EtOH, 99%; (d) 55,
MeOTf, DTBP, DCM, Et2O, 78%; (e) 1. NH2CH2CH2NH2, EtOH, reflux; 2. Na/NH3, THF; 3. Ac2O, pyridine, DMAP; 4. NaOMe, MeOH (30%, four steps).
This pivotal finding paved the way for a much more efficient
and straightforward approach to the preparation of N-linked
glycans. Not only is the need for a moisture-sensitive and
problematic thiolysis step obviated, but both isomeric sul-
fonamides can be utilized in the hydrolysis, greatly simplify-
ing the transformation and allowing higher throughput of
material.
Hemiacetal 72 was subjected to reduction with sodium in
liquid ammonia (Scheme 12),32 and happily, the reducing
end hemiacetal proved sufficiently robust; the fully depro-
tected material, 73, was isolated in 57% yield over two steps,
following partial reacetylation and reverse-phase column
chromatography. The reducing alcohol was then converted
into anomeric amine 74 under Kochetkov amination condi-
tions,16 and the latter was then coupled with pentapeptide
48 to furnish the N-linked glycopeptide 3, presenting the
H-type 2 blood group specificity.
We were eager to apply this novel approach to our pentade-
casaccharide system (Scheme 11). Thus, the phthalimides in
67 were first converted into acetamides (cf. 71). Then, the
glycal was subjected to iodosulfonamidation, followed by
basic hydrolysis of the resulting product mixture, to afford
pentadecasaccharide 72. At this point, the stage was set for
the critical global deprotection.
The ability to gain access through chemical synthesis to such
an advanced N-linked glycopeptide in a homogeneous state
provided a unique opportunity to investigate not only its gly-
coarchitecture but also the biological implications of the

4964
Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
BnO
BnO
OBn
O
BnO
O
OBn
O
BnO
BnO
O
O
O
O
BnO
O
BnO
O
BnO
PhthN
PhthN
BnO
O
OBn
O
OBn
BnO
BnO
BnO
BnO
BnO
OAc
O
O
O
BnO
a
BnO
PMBO
I
O
b
67
O
O
O
BnO
BnO
BnO
PhSO2N
H
BnO
OBn
PhSO2N
H
O
BnO
OBn
O
PhthN
68
BnO
PhthN
O
O
O
BnO
O
O
BnO
O
O
O
O
OBn
BnO
BnO
BnO
OBn
BnO
BnO
OBn
O
BnO
O
OBn
O
BnO
BnO
O
BnO
BnO
BnO
BnO
O
O
O
BnO
O
O
BnO
O
PhthN
BnO
PhthN
BnO
O
OBn
OBn
BnO
BnO
OAc
O
O
BnO
BnO
PMBO
O
O
O
R
O
O
BnO
BnO
PhSO2N
H
BnO
PhSO2N
BnO
OBn
O
H
BnO
OBn
O
PhthN
O
69 R = SEt
70 R = OH
BnO
PhthN
O
O
O
BnO
O
O
BnO
O
O
O
OBn
BnO
BnO
BnO
OBn
BnO
Scheme 10. Functionalization of the terminal glycal: (a) PhSO2NH2, IDCP; (b) EtSH, LiHMDS, DMF, 70 only, 40% (two steps).
Table 1. Dissolving metal reduction of glycal-derived 1-hydroxy sugars
Deprotection substrate
Peracetylated substrate
Time (min)
60
Yielda (%)
64
BnO
O
AcO
O
BnO
AcO
OH
OH
OAc
BnO
AcO
PhSO2N
AcN
H
H
BnO
AcO
O
O
BnO
AcO
20
60
65
76
OAc
O
BnO
AcO
OH
OAc
BnO
AcO
AcO
OBn
OAc
O
O BnO
AcO
O
BnO
O
O
OH
OH
OH
OAc
BnO
AcO
BnO
AcO
PhSO2N
H
AcN
H
BnO
BnO
AcO
AcO
OBn
O
OAc
O
BnO
AcO
O
O
O
O
60
20
72
79
OAc
OAc
BnO
AcO
BnO
AcO
AcN
H
AcN
H
BnO
BnO
AcO
AcO
OBn
O
OAc
O
BnO
O
AcO
O
O
O
BnO
AcO
BnO
AcO
OH
OAc
Reactions were performed by the addition of the saccharide in THF to a stirred solution of Na (6 equiv perbenzyl group) in NH3 at ꢁ78 ꢀC. After quenching and
workup, the crude product was acetylated for analysis.
Isolated yield following column chromatography.
a

Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
4965
BnO
BnO
OBn
O
BnO
O
OBn
O
BnO
O
BnO
O
O
O
BnO
O
O
BnO
AcN
H
BnO
AcN
H
BnO
O
OBn
O
OBn
BnO
BnO
BnO
BnO
BnO
OAc
O
a
O
BnO
O
67
PMBO
O
BnO
O
O
O
BnO
BnO
BnO
PhSO2N
Ac
BnO
OBn
O
BnO
OBn
O
AcHN
AcHN
O
BnO
O
BnO
O
71
O
BnO
O
O
O
O
OBn
BnO
O
OBn
BnO
BnO
BnO
b
BnO
BnO
OBn
O
BnO
O
OBn
O
BnO
O
O
BnO
O
O
BnO
O
O
BnO
AcN
H
BnO
AcN
H
BnO
O
OBn
O
OBn
BnO
BnO
BnO
BnO
BnO
OAc
O
O
BnO
O
PMBO
O
BnO
O
O
O
BnO
OH
BnO
BnO
PhSO2N
Ac
PhSO2N
H
BnO
OBn
O
BnO
OBn
O
AcHN
AcHN
O
BnO
O
BnO
O
72
O
BnO
O
O
O
O
OBn
BnO
O
OBn
BnO
BnO
BnO
Scheme 11. Upgrading of the terminal glycal to a hemiacetal: (a) 1. NH2CH2CH2NH2, EtOH, 2. Ac2O, pyridine, DMAP, 85% (two steps); (b) 1. IDCP,
PhSO2NH2, DCM, 74%; 2. LiHMDS, AgOTf, THF, H2O, 63% or aq K2CO3, THF, 60%.
attached H-type 2 specificity. Indeed, the 1H NMR spectrum
of 3 (Fig. 4) is extremely well resolved, indicative of a high
degree of structural order.25 Moreover, the functionality of
the H-type 2 blood group determinants was confirmed in
an enzyme-linked immunosorbent assay (ELISA) where
glycopeptide 3 reacted with an antibody against the H-type
HO
O
HO
O
a
b
O
O
72
OH
NH2
HO
HO
AcN
H
AcN
H
73
74
OH
OH
NH2
O
O
O
O
H
H
N
N
c
N
N
N
H
H
H
H
O
CH3
O
O
48
HO
OH
O
HO
OH
O
HO
O
HO
HO
O
O
O
O
HO
O
O
HO
AcN
H
OH
AcN
H
HO
O
OH
O
OH
HO
HO
HO
HO
HO
OH
O
O
HO
HO
O
HO
O
O
O
O
HO
NH
HO
HO
AcN
H
AcN
H
HO
OH
O
OH
HO
O
OH
O
O
O
O
H
N
H
AcHN
N
NH2
AcHN
O
HO
O
O
HO
O
N
N
N
H
O
HO
H
O
H
H
O
3
O
O
CH3
O
O
O
OH
HO
HO
HO
OH
HO
Scheme 12. Stereocontrolled synthesis of a b-N-linked 15-mer glycopeptide containing the H-type 2 blood group determinants: (a) 1. Na/NH3, THF; 2. Ac2O,
MeOH, 57% (two steps); (b) NH4HCO3, H2O, >99%; (c) 48, HOBt, HBTU, DIPEA, DMSO, 20%.

4966
Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
8.6
8.4
8.2
ppm
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0 ppm
Figure 4. The 1H NMR spectrum (800 MHz) of 3 in D2O at 20 ꢀC and pH 3.7 (phosphate buffer). The inset shows the secondary NH signals of amides from the
peptide backbone, side chain, and GlcNAc sites when the spectrum is taken in H2O at 5 ꢀC and pH 3.7 (phosphate buffer).
2 determinant (Fig. 5). This antibody is highly specific for
the H-type 2 blood group determinant, and does not react
with related structures, such as H-type 1, Lea, or Lex.33,34
neous cleavage of multiple benzyl groups was appropriate,
even in this most difficult context (cf. 72/73). Happily
the Kotchetkov–Lansbury amination–aspartylation was
also appropriate (cf. 73/3).
500000
400000
300000
200000
100000
0
With the ability to reach reasonable quantities of homoge-
neous structures containing this level of complexity in the
laboratory come new questions. For instance, is it possible
to develop methodology wherein complex glycopeptides
can be ligated to create even more biorelevant structures?
If so, can the chemistry be conducted in such a fashion
that it would allow for the conservation of the fragile glyco-
sidic linkages? One could then consider even more challeng-
ing systems as targets for chemical synthesis. One of the
most exciting dimensions of synthetic chemistry, in our
view, is its capacity to deal with still more biopertinent struc-
tures which might illuminate Nature’s ways and even, occa-
sionally serve to modulate these processes in constructive
directions. Having reached the H-type 2 human blood group
structural context by chemical synthesis, we were led to
wonder if a system of the complexity of, say, erythropoie-
tin35 might be accessible via suitable advances in methodol-
ogy. It is toward such goals that we have recently directed
considerable attention.36
256
16
1
6.3 x 10-2
3.9 x 10-3
Antigen / µg carbohydrate mL-1
Figure 5. Reactivity of glycopeptide 3 (-), H-type 2 active mucin (:) as
a positive control, and Lex/Lea active mucin (;) as negative control with the
antibody against H-type 2 determinants (mAbSA) as determined with an
ELISA. The mucin preparations have been previously reported.33
3. Conclusion
In conclusion, the goals of our investigation were substan-
tially met. The logic of glycal assembly, honed over a decade,
proved equal to the task of synthesizing the complex oligo-
saccharide 71. The glycal olefin proved susceptible to our
sulfonamidohydroxylation methodology. More importantly,
the powerful Birch reduction methodology for the simulta-
4. Experimental
4.1. General
ꢀ
All glassware was dried in an oven at 140 C before use. All
experiments were conducted under an atmosphere of dry

Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
4967
argon unless indicated otherwise. Solvents were dried as fol-
lows: methylene chloride, diethyl ether, tetrahydrofuran
(THF), benzene, and toluene were obtained from a dry
solvent system (alumina) and used without further drying.
Pyridine and triethylamine were freshly distilled from
CaH2. All NMR spectra were recorded on a Bruker model
AMX-400 (1H: 400 MHz, 13C: 100 MHz) or a Bruker model
DRX-500 (1H: 500 MHz, 13C: 125 MHz) NMR spectrome-
ter. Chemical shifts are reported in parts per million (ppm)
from internal tetramethylsilane (1H NMR spectra,
d 0.00 ppm) or the residual solvent signal of CDCl3 (13C
NMR spectra, d 77.23 ppm). Infrared spectra were taken
on a Perkin Elmer 1600 Series FTIR spectrometer using
thin film deposition on polished NaCl plates. Peaks are re-
ported in wavenumbers (cmꢁ1). Low and high-resolution
mass spectra were obtained from a PE Sciex API 100 instru-
ment under EI mode, and are reported in units of m/z. Col-
umn chromatography (low-pressure chromatography) was
performed with E. Merck silica gel 60 (40–63 mesh). Thin
layer chromatography (TLC) was carried out on Merck silica
gel 60 F-254 glass-backed plates. TLC visualization was
done with a 254 nm UV lamp and potassium permanganate
or phosphomolybdic acid staining solution. All chemicals
were purchased from Aldrich Chemical Co. and used as re-
ceived. While we cannot rule out the possibility of anomeric
mixtures during glycosylation reactions, we were unable to
observe such mixtures and the yields reported for these reac-
tions are for the pure anomer listed.
then stirred for another 20 min. The reaction was quenched
with H2O (20 mL) and extracted with ether (3ꢂ300 mL).
The combined extracts were washed with saturated aq
NaHCO3 (200 mL) and brine (300 mL) then dried
(MgSO4) and concentrated. The crude material was purified
by silica gel chromatography (hexane/EtOAc¼6.5:1) to af-
ford the desired 3,4,6-tribenzyl thioethyl mannoside
(7.05 g, 14.2 mmol, 76%) as a white foam.
4.1.3. Synthesis of 2-tert-butyldimethylsilyl-3,4,6-tri-
benzyl-1-thioethyl-b-mannoside (24). To a solution of 3,4,6-
tribenzyl-1-thioethyl-b-mannoside (500 mg, 1.01 mmol) in
CH2Cl2 (5 mL) was added triethylamine (2.81 mL,
20.2ꢀmmol) and TBSOTf (464 mL, 2.02 mmol) drop wise
at 0 C. The reaction was slowly warmed to room tempera-
ture and stirred for 3 h. The reaction was diluted with EtOAc
(100 mL), washed with saturated aq NaHCO3 and brine then
dried (MgSO4), and concentrated in vacuo. Purification by
silica column chromatography (hexane/EtOAc¼10:1)
yielded 24 (570 mg, 0.94 mmol, 93%). [a]2D5 ꢁ36.1
(c¼4.09, CH2Cl2); IR (thin film): 3087, 2926, 2916, 1496,
1
1463, 1362, 1251, 1102, 1027, 966, 834 cmꢁ1; H NMR
(400 MHz, CDCl3): d¼7.40–7.21 (m, 15H), 4.90 (d,
J¼8.8 Hz, 1H), 4.81 (d, J¼11.8 Hz, 1H), 4.69 (d,
J¼4.5 Hz, 1H), 4.62 (d, J¼4.7 Hz, 1H), 4.58 (d, J¼5.3 Hz,
1H), 4.51 (d, J¼4.0 Hz, 1H), 4.49 (s, 1H), 4.22 (d,
J¼2.4 Hz, 1H), 3.93 (t, J¼9.5 Hz, 1H), 3.75–3.68 (m, 2H),
3.52–3.48 (m, 2H), 2.75–2.72 (m, 1H), 1.33 (t, J¼7.3 Hz,
3H), 0.98–0.96 (m, 9H), 0.20–0.13 (m, 6H); 13C NMR
(100 MHz, CDCl3): d¼137.3, 136.9, 136.8, 127.0, 126.9,
126.8, 126.8, 126.4, 126.3, 126.2, 125.9, 83.9, 82.7, 78.8,
73.7, 73.1, 71.8, 71.3, 71.1, 68.1, 24.8, 24.3, 17.3, 13.8,
ꢁ4.9, ꢁ5.4, ꢁ5.7; HRMS calcd for C35H48O5S1Si1Na:
631.2889, found: 631.2883.
4.1.1. Improved synthesis of 4-p-methoxybenzylglucal
(21). To a solution of 4,6-p-methoxybenzylidine glucal15
ꢀ
(20) (6.00 g, 23 mmol) in CH2Cl2 (20 mL) at ꢁ78 C was
added Dibal-H (1.5 M solution in toluene, 58 mL,
87 mmol). After removing the cooling bath, the resulting
mixture was stirred at room temperature for 4 h and then di-
luted with CH2Cl2 (200 mL). The mixture was treated with
saturated aq KNa-tartrate (100 mL) and was stirred for
15 h. The organic layer was separated, washed with brine,
and dried over Na2SO4 and then concentrated in vacuo.
The residue was purified by silica gel chromatography (hex-
ane/EtOAc¼1:1) to afford 21 (4.5 g, 17 mmol, 74%) as
a white waxy solid. [a]D25 15.3 (c¼2.86; CH2Cl2); IR (thin
film): 3285, 2955, 2933, 2836, 1649, 1613, 1514, 1232,
4.1.4. Synthesis of 2-tert-butyldimethylsilyl-3,4,6-tri-
benzyl-1-thioethyl-a-mannoside (27). 2-Acetyl-3,4,6-
tribenzyl-1-thioethyl-a-mannoside (14.1 g, 26 mmol) was
dissolved in MeOH (50 mL) then treated with NaOMe
(25% solution in MeOH, 0.6 mL, 2.62 mmol). The reaction
was stirred for 12 h and then was neutralized with Amberlyst
15 and filtered. The resin was washed with MeOH
(3ꢂ50 mL). The combined filtrates were concentrated under
reduced pressure and azeotroped with benzene (3ꢂ30 mL)
then dried under high vacuum to afford the free alcohol
(12.8 g). The crude alcohol was dissolved in CH2Cl2
(50 mL) and Et3N (15 mL, 107.6 mmol) then cooled to
1173, 1086, 952, 814, 761 cmꢁ1 1H NMR (500 MHz,
;
CDCl3): d¼7.26 (d, J¼8.6 Hz, 2H), 6.85 (d, J¼8.6 Hz,
2H), 6.30 (dd, J¼1.3, 6.0 Hz, 1H), 4.75–4.65 (m, 3H),
4.30 (br d, J¼5.4 Hz, 1H), 3.88–3.77 (m, 3H), 3.75 (s,
3H), 3.57 (dd, J¼6.7, 8.8 Hz, 1H), 2.79 (br s, 1H), 2.70 (br
s, 1H); 13C NMR (125 MHz, CDCl3): d¼159.4, 144.1,
130.2, 129.7, 114.0, 103.3, 77.4, 76.6, 73.4, 71.6, 68.9,
61.7, 55.2.
ꢀ
0 C. TBSOTf (7.83 mL, 34 mmol) was added and the
mixture was warmed to room temperature over 4 h then di-
luted with EtOAc (500 mL). The solution was washed
with saturated aq NaHCO3 and brine then dried (Na2SO4)
and concentrated in vacuo. The residue was purified by silica
gel chromatography (hexane/EtOAc¼20:1) to afford 27
(14.7 g, 24.2 mmol, 93% over two steps) as a clear, colorless
4.1.2. Synthesis of 3,4,6-tribenzyl-1-thioethyl-b-manno-
side. 3,4,6-Tribenzyl-1-thioethyl-b-glucopyranoside12 (23)
(9.29 g, 18.7 mmol) was treated with DMSO/Ac2O
(100 mL/50 mL) at room temperature for 3 d. The reaction
mixture was then diluted with diethyl ether (1 L) and washed
with H2O (5ꢂ200 mL), saturated aq Na2CO3 (3ꢂ200 mL),
and brine. The crude ketone was dried over MgSO4 and con-
centrated in vacuo. The residue was dissolvedꢀin CH2Cl2
(60 mL) and MeOH (60 mL) then cooled to 0 C. NaBH4
(2.13 g, 37.8 mmol) was added in several portions and the
reaction mixture was allowed to warm to room temperature
1
oil. H NMR (500 MHz, CDCl3): d¼7.20–7.40 (m, 15H),
5.10 (d, J¼1.2 Hz, H-2), 4.72 (d, J¼10.8 Hz, 1H), 4.60
(d, J¼11.7 Hz, 1H), 4.58 (d, J¼12.3 Hz, 1H), 4.50 (d,
J¼11.7 Hz, 1H), 4.40 (d, 12.3 Hz, 1H), 4.38 (d,
J¼10.8 Hz, 1H), 4.10 (s, 1H), 4.07 (m, 1H), 3.95
(dd, J¼9.4, 9.4 Hz, 1H), 3.60–3.80 (m, 3H), 2.52 (m, 2H),
1.25 (t, J¼7.5 Hz, 3H), 0.85 (s, 9H), 0.09, 0.00, ꢁ0.02 (3s,
12H). HRMS calcd for C35H48O5S1Si1Na: 631.2889, found:
631.2878.

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Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
4.1.5. Synthesis of 3,4,6-tribenzyl-2-p-toloyl-1-thioethyl-
a-mannoside (28). To a solution of 3,4,6-tribenzyl-1-thio-
ethyl-a-mannoside (8.0 g, 16 mmol) in CH2Cl2 (50 mL)
was added triethylamine (25 mL, 18 mmol) followed by p-
and brine then dried (Na2SO4) and concentrated. The residue
was purified over silica gel (hexane/EtOAc¼20:1, then 10:1)
to afford 30 (2.3 g, 1.69 mmol, 95%) as a thick syrup. [a]D25
24.4 (c¼0.6, CHCl3); IR (thin film) 3030, 2926, 2855, 1648,
1
1514, 1454, 1380, 1249, 1043, 835, 777 cmꢁ1; H NMR
ꢀ
toluoyl chloride (8.5 mL, 63.7 mmol) at 0 C. The resulting
mixture was warmed to room temperature and stirred for 2 d.
Saturated aq NaHCO3 (50 mL) was added and the mixture
was stirred vigorously for 15 h then diluted with EtOAc
(500 mL). After separation, the organic extract was washed
with saturated aq NaHCO3 and brine then dried (Na2SO4)
and concentrated invacuo. Purification over silica gel (hexane/
EtOAc¼20:1, then 10:1) yielded 28 (7.4 g, 12.1 mmol,
75%) as yellowish syrup. 1H NMR (500 MHz, CDCl3):
d¼7.89–7.91 (m, 2H), 7.19–7.33 (m, 15H), 7.08–7.10 (m,
2H), 5.65 (dd, J¼1.4, 3.0 Hz, 1H), 5.38 (d, J¼1.4 Hz, 1H),
4.79, (d, J¼10.5 Hz, 1H), 4.71, (d, J¼11.2 Hz, 1H), 4.69
(d, J¼11.9 Hz, 1H), 4.47 (d, J¼11.2 Hz, 1H), 4.44 (d,
J¼11.9 Hz, 1H), 4.40 (d, J¼10.5 Hz, 1H), 4.14 (ddd,
J¼1.6, 3.4, 9.7 Hz, 1H), 4.07 (dd, J¼9.2, 9.7 Hz, 1H),
3.93 (dd, J¼3.0, 9.2 Hz, 1H), 3.87 (dd, J¼3.4, 10.6 Hz,
1H), 3.69 (dd, 1.6, 10.6 Hz, 1H), 2.50–2.64 (m, 2H), 2.34
(s, 3H), 1.21 (t, J¼7.4 Hz, 3 Hz); 13C NMR (125 MHz,
CDCl3): d¼166.1, 144.2, 138.7, 138.7, 138.1, 131.2,
130.3, 129.4, 128.6, 128.4, 128.3, 128.0, 127.9, 127.9,
127.8, 127.5, 126.5, 82.9, 79.0, 76.0, 74.8, 73.7, 72.3,
71.8, 70.9, 69.3, 25.9, 22.0, 15.3.
(400 MHz, CDCl3) d¼7.40–7.12 (m, 32H), 6.79 (dd,
J¼2.0, 6.4 Hz, 2H), 6.21 (dd, J¼1.2, 6.4 Hz, 1H), 5.01
(dd, J¼2.2, 6.1 Hz, 1H), 4.94 (d, J¼1.9 Hz, 1H), 4.84 (d,
J¼10.8 Hz, 2H), 4.79 (d, J¼2.0 Hz, 1H), 4.78 (d,
J¼11.7 Hz, 1H), 4.68–4.60 (m, 6H), 4.56–4.45 (dd, J¼3.0,
11.9 Hz, 4H), 4.51 (dd, J¼3.6, 10.8 Hz, 1H), 4.36 (dd,
J¼1.7, 7.1 Hz, 1H), 4.13 (t, J¼2.4 Hz, 1H), 4.02–3.82 (m,
8H), 3.78–3.63 (m, 7H), 3.73 (s, 3H), 0.89 (b, 18H), 0.10
(s, 3H), 0.06 (s, 3H), 0.05 (s, 3H), 0.01 (s, 3H); 13C NMR
(100 MHz, CDCl3): d¼159.3, 144.2, 138.6, 138.5, 138.4,
130.1, 129.2, 128.3, 128.2, 128.2, 128.2, 128.2, 128.1,
127.6, 127.6, 127.4, 127.4, 127.3, 127.2, 113.9, 102.6,
102.3, 101.2, 80.0, 79.9, 79.2, 75.1, 75.0, 74.9, 74.7, 74.0,
73.1, 73.0, 72.6, 72.4, 72.1, 70.4, 69.6, 69.4, 69.2, 65.7,
55.2, 25.8, 25.7, 18.1, 0.0, ꢁ4.5, ꢁ4.8, ꢁ4.8; HRMS calcd
for C80H102O15Si2Na: 1381.6650, found: 1381.6670.
Method B (TBS protecting group): Thiodonor 27 (8 g,
13 mmol) and glucal acceptor 21 (1.15 g, 4.38 mmol) were
mixed and azeotroped with benzene (3ꢂ30 mL), and then
further dried under high vacuum. To the residue was added
CH2Cl2 (100 mL) followed by 2,6-di-tert-butylpyridine
˚
4.1.6. Synthesis of disilyl dimannosyl trisaccharide glycal
30. Method A (p-toluoyl protecting group): Thiodonor 28
(7.4 g 12.1 mmol) and glucal acceptor 21 (1.0 g, 4 mmol)
were mixed and azeotroped with toluene (3ꢂ40 mL). The
residue was dissolved in CH2Cl2 (20 mL) and Et2O
(20 mL) followed by the addition of 2,6-di-tert-butylpyridine
(14.7 mL, 65 mmol) and freshly dried 4 A molecular sieves
(10 g). The slurryꢀwas stirred at room temperature for 30 min
then cooled to 0 C and MeOTf (5.94 mL, 52.5 mmol) was
added. The reaction was slowly warmed to room tempera-
ture over 4 h with vigorous stirring then quenched with sat-
urated aq NaHCO3 (10 mL) and diluted with EtOAc
(500 mL). The mixture was filtered through a pad of Celite,
and the filtrate was washed with saturated aq NaHCO3
(50 mL) and brine (50 mL) then dried (Na2SO4) and concen-
trated in vacuo. The residue was purified over silica gel
(hexane/EtOAc¼1:0, 10:1, then 5:1) to yield 30 (3.2 g,
2.36 mmol, 53%) as a thick syrup.
˚
(13.45 mL, 60 mmol) and freshly dried 4 A molecular sieves
(10 g). The resulting slurry was stirred for 30 min at room
ꢀ
temperature then cooled to 0 C. MeOTf (5.42 mL,
48 mmol) was added and the mixture was slowly warmed
to room temperature and stirred for 15 h at which point
Et3N (5 mL, 35.8 mmol) was added. After an additional
15 min of stirring, the solution was filtered through a pad
of Celite and concentrated. The residue was purified over sil-
ica gel (hexane/EtOAc¼5:1) to yield 29 (3.3 g, 2.41 mmol,
63%) as a thick, colorless oil. 1H NMR (500 MHz, CDCl3):
d¼8.05 (m, 4H), 7.20–7.50 (m, 23H), 6.80 (d, J¼8.7 Hz,
2H), 6.30 (d, J¼6.4 Hz, 1H), 5.70 (d, J¼1.0 Hz, 1H), 5.60
(d, J¼2.4 Hz, 1H), 5.22 (d, J¼1.7 Hz, 1H), 5.05 (d,
J¼1.6 Hz, 1H), 4.50–5.00 (m, 10H), 4.50–4.70 (m, 10H),
3.70–4.30 (m, 7H), 3.58 (s, 3H), 2.45 (s, 3H), 2.43 (s, 3H).
4.1.7. Synthesis of dimannosyl trisaccharide alcohol thio-
glycoside 32. To a solution of 30 (1.0 g, 0.73 mmol) in
˚
CH2Cl2 (10 mL) was added 4 A MS (flame-dried, 1 g).
The resulting slurry was ꢀstirred at room temperature for
30 min then cooled to 0 C. Dimethyldioxirane (0.072 M
in acetone, 12.2 mL, 0.876 mmol) was added very slowly
(important) under vigorous stirring over 1 h. After the addi-
tion was complete, the mixture was stirred for 10 min and the
volatiles were removed under a stream of dry N2. The crude
epoxide (31) was azeotroped with benzene (ꢂ3) then dis-
The trisaccharide glycal 29 (3.3 g, 2.4 mmol) was dissolved
in MeOH (20 mL) and toluene (5 mL) and NaOMe (25%
solution in MeOH, 0.2 mL, 0.87 mmol) was added. The
mixture was stirred at room temperature for 15 h then con-
centrated in vacuo. The residue was purified over silica gel
(hexane/EtOAc¼2:1, then 1:1) to afford the dihydroxylated
glycal (2.2 g, 1.94 mmol, 77%) as a thick syrup.
ꢀ
solved in CH2Cl2 (5 mL) and cooled to ꢁ78 C. Ethanethiol
(2.5 mL, 33.75 mmol) and trifluoroacetic anhydride (10 mL,
0.07 mmol) were added and the reaction was stirred for
10 min then quenched with Et3N (100 mL, 0.72 mmol). Af-
ter removing the volatiles under a N2 stream, the residue
can be azeotroped with benzene (ꢂ2) for direct acylation
or can be purified over silica gel (hexane/EtOAc¼5:1) to af-
ford the thioglycoside 32 (0.80 g, 0.58 mmol, 76%) as a thick
syrup. [a]2D4 12.8 (c¼0.7, CHCl3); IR (thin film): 3472, 2927,
2856, 1613, 1514, 1454, 1360, 1250, 1137, 1092, 1049, 978,
835, 777 cmꢁ1; 1H NMR (400 MHz, CDCl3): d¼7.40–7.10
(m, 32H), 6.80 (d, J¼8.4 Hz, 1H), 4.90 (d, J¼1.8 Hz, 1H),
4.84 (d, J¼10.8 Hz, 2H), 4.79 (d, J¼11.8 Hz, 1H), 4.65
The dihydroxy glycal (2.0 g, 1.76 mmoꢀl) was dissolved in
CH2Cl2 (20 mL) and cooled to 0 C. Triethylamine
(1.22 mL, 8.75 mmol) and TBSOTf (1.2 mL, 5.27 mmol)
were added and the resulting mixture was stirred at room
temperature for 3 h then diluted with EtOAc (200 mL).
The organic layer was washed with saturated aq NaHCO3

Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
4969
(d, J¼11.0 Hz, 2H), 4.58–4.45 (m, 9H), 4.39 (d, J¼10.6 Hz,
1H), 4.34 (d, J¼9.6 Hz, 1H), 4.08 (dd, J¼2.2 Hz, 2H), 4.04–
4.01 (m, 1H), 3.91 (dd, J¼9.0, 18.0 Hz, 2H), 3.84 (d,
J¼2.4 Hz, 1H), 3.84–3.66 (m, 11H), 3.59 (t, J¼8.1 Hz,
1H), 3.43–3.32 (m, 3H), 2.75–2.60 (m, 2H), 1.23 (t,
J¼7.6 Hz, 3H), 0.90 (s, 9H), 0.88 (s, 9H), 0.10 (s, 3H),
0.07 (s, 3H), 0.05 (s, 3H), 0.01 (s, 3H); 13C NMR
(100 MHz, CDCl3): d¼159.3, 138.6, 138.6, 138.2, 138.2,
138.1, 128.8, 128.3, 128.3, 128.2, 128.1, 128.0, 128.0,
127.7, 127.6, 127.5, 127.5, 127.4, 127.4, 127.3, 127.2,
114.0, 102.8, 100.9, 90.8, 84.8, 78.1, 76.3, 75.1, 74.9,
74.9, 74.6, 73.2, 72.9, 72.7, 72.1, 72.0, 70.1, 69.4, 69.3,
69.1, 65.7, 55.2, 18.1, 15.0, ꢁ4.5, ꢁ4.5, ꢁ4.7, ꢁ4.9;
FAB(+)MS: 1460, 1439, 1357, 963; HRMS calcd for
C82H108O16SSi2Na: 1459.6790, found: 1459.6760.
4.1.10. Synthesis of pentasaccharide glycal acetate 35.
Trisaccharide donor 33 (1.03 g, 0.695 mmol) and disaccha-
ride acceptor 19 (561 mg, 0.695 mmol) were combined
˚
and azeotroped with benzene (ꢂ2). Freshly activated 4 A
molecular sieves (4.0 g) were added, followed by CH2Cl2
(8 mL) and 2,6-di-tert-butylpyridine (1.09 mL, 4.9 mmol)
and the mixture was stirred for 45ꢀmin at room temperature.
The reaction was cooled to ꢁ10 C and MeOTf (0.47 mL,
4.2 mmol) was slowly added. The reaction mixture was
ꢀ
ꢀ
stiꢀrred at ꢁ8 C for 10 h, at ꢁ5 C for 6 h and finally at
5 C for 6 h then quenched with Et3N (2.0 mL,
14.3 mmol), filtered through a plug of silica, washed with
NaHCO3, and extracted with EtOAc. The combined organic
extracts were washed with water and brine, dried (MgSO4),
and concentrated in vacuo. Purification by silica gel chroma-
tography (hexane/EtOAc¼3:1) yielded 35 (994 mg,
0.45 mmol, 64%) as a white foam. [a]2D4 ꢁ5.3 (c¼1.31,
CH2Cl2); IR (thin film) 2247, 1650, 1612, 1586, 1571,
4.1.8. Synthesis of dimannosyl trisaccharide acetate thio-
glycoside 33. To a room temperature solution of 32 (3.01 g,
2.09 mmol) in CH2Cl2 was added Ac2O (1.07 g,
10.4 mmol), Et3N (2.11 g, 20.9 mmol), and a catalytic
amount of DMAP. After stirring for 2 h the reaction was con-
centrated in vacuo and the remaining residue was purified di-
rectly by silica gel chromatography to afford 33 (2.81 g,
1.90 mmol, 91%). [a]D24 24.6 (c¼1.0, CHCl3); IR (thin
film): 2927, 2855, 1750, 1613, 1514, 1454, 1361, 1249,
1
1514, 1498, 1454, 1361, 1249, 910, 836 cmꢁ1; H NMR
(400 MHz, CDCl3): d¼7.87 (d, J¼7.7 Hz, 2H), 7.45–7.19
(m, 45H), 6.76 (d, J¼8.8 Hz, 2H), 6.34 (d, J¼6.0 Hz, 1H),
5.05 (br d, J¼1.6 Hz, 1H), 4.95–4.77 (m, 6H), 4.75–4.35
(m, 24H), 4.23 (d, J¼6.2 Hz, 1H), 4.16–3.95 (m, 7H),
3.90–3.70 (m, 14H), 3.75 (s, 3H), 3.70–3.35 (m, 14H),
3.24–3.19 (m, 1H), 1.98 (s, 3H), 0.94 (s, 9H), 0.88 (s, 9H),
0.10 (s, 3H), 0.06 (s, 6H), ꢁ0.03 (s, 3H); 13C NMR
(100 MHz, CDCl3) d¼170.3, 159.0, 144.4, 141.8, 138.8,
138.7, 138.7, 138.6, 138.6, 138.5, 138.4, 137.9, 137.9,
137.8, 133.5, 128.6, 128.4, 128.4, 128.3, 128.3, 128.2,
128.1, 128.1, 128.1, 128.1, 128.0, 127.9, 127.7, 127.6,
127.4, 127.4, 127.3, 127.3, 127.2, 127.2, 127.2, 113.8,
101.6, 101.3, 100.0, 99.8, 99.4, 79.8, 79.5, 77.9, 75.4,
75.0, 74.7, 74.5, 74.3, 74.2, 74.0, 73.6, 73.4, 73.3, 73.3,
73.2, 73.0, 72.9, 72.8, 72.5, 72.5, 72.2, 72.1, 71.7, 70.2,
69.9, 69.1, 69.0, 67.7, 65.5, 56.2, 55.2, 25.7, 25.6, 21.1,
18.1, ꢁ4.4, ꢁ4.5, ꢁ4.6, ꢁ4.7, ꢁ5.1; HRMS calcd for
C128H153NO27SSi2Na: 2246.9786, found: 2246.9840.
1
1225, 1050, 835, 777 cmꢁ1; H NMR (400 MHz, CDCl3):
d¼7.40–7.05 (m, 30H), 6.75 (d, J¼8.4 Hz, 2H), 5.06 (br s,
1H), 4.98–4.72 (m, 6H), 4.70–4.40 (m, 8H), 4.29 (d,
J¼8.5 Hz, 1H), 4.08–4.06 (br m, 3H), 3.98–3.55 (m, 14H),
3.68 (s, 3H), 3.48–3.42 (m, 1H), 3.39–3.35 (m, 1H), 2.65–
2.60 (m, 1H), 2.23 (s, 3H), 0.91 (s, 9H), 0.84 (s, 9H), 0.12
(s, 3H), 0.08 (s, 3H), 0.02 (s, 3H), ꢁ0.06 (s, 3H); 13C
NMR (100 MHz, CDCl3): d¼169.9, 166.3, 159.1, 138.9,
138.7, 138.6, 138.5, 138.4, 129.7, 128.3, 128.2, 128.2,
128.1, 128.1, 128.0, 127.7, 127.5, 127.3, 127.3, 127.2,
127.2, 127.2, 113.9, 102.1, 101.2, 83.5, 79.9, 79.5, 78.4,
74.9, 74.7, 74.6, 74.0, 73.2, 73.0, 72.9, 72.3, 72.2, 72.0,
71.1, 69.4, 69.2, 65.5, 60.3, 55.2, 25.7, 25.7, 24.0, 22.1,
21.2, 18.1, 18.1, 14.8, ꢁ4.5, ꢁ4.5, ꢁ4.8, ꢁ5.1; HRMS calcd
for C84H110O17SSi2Na: 1501.6900, found: 1501.6900.
4.1.11. Synthesis of pentasaccharide glycal benzoate 36.
Trisaccharide donor 34 (2.2 g, 1.45 mmol) and disaccharide
acceptor 19 (1.5 g, 1.85 mmol, 1.3 equiv) were combined
and azeotroped with benzene (3ꢂ20 mL). The residue was
4.1.9. Synthesis of trisaccharide benzoate thioglycoside
34. To a solution of 32 (3.28 g, 2.34 mmol) and pyridine
(5 mL, 61.8 mmol) in CH2Cl2 (50 mL) was added benzoyl
chloride (1.08 mL, 9.36 mmol) and the resulting mixture
was stirred at room temperature for 4 d. At that point, prop-
anol (1 mL) was added and stirring was continued for an ad-
ditional hour. The mixture was diluted with EtOAc (200 mL)
and washed with saturated aq NaHCO3 and brine. The or-
ganic layer was dried over Na2SO4 and then concentrated
in vacuo. The residue was purified over silica gel (hexane/
EtOAc¼3:1) to afford 34 (3.2 g, 2.11 mmol, 90%) as a white
˚
dissolved in dry CH2Cl2 (20 mL) then activated 4 A molec-
ular sieves (5 g) and 2,6-di-tert-butylpyridine (1.30 mL,
5.78 mmol) were added. The slurry was stirred at room tem-
ꢀ
3.53 mmol) was added and the resulting mixture was stirred
at a temperature between 0 C and room temperature for 2 d
perature for 30 min then cooled to 0 C. MeOTf (0.40 mL,
ꢀ
then quenched by the addition of saturated aq NaHCO3
(1 mL) and diluted with EtOAc (200 mL). After filtration
through a short pad of Celite, the filtrate was washed with
saturated aq NaHCO3 (20 mL) and brine (20 mL) then dried
(Na2SO4) and concentrated in vacuo. The residue was puri-
fied over silica gel (hexane/EtOAc¼3:1) to afford 36 (2.36 g,
1.03 mmol, 71%) as a white foam. Rf 0.5 (hexane/
EtOAc¼3:1), [a]2D5 ꢁ5.0 (c¼1.0, CHCl3); IR (thin film):
3468, 3030, 2933, 2858, 1651, 1496, 1092 cmꢁ1; 1H NMR
(500 MHz, CDCl3): d¼7.70 (d, J¼8.0 Hz, 2H), 7.20–7.50
(m, 62H), 6.78 (d, J¼8.0 Hz, 1H), 6.40 (d, J¼6.2 Hz, 1H),
4.40–4.59 (m, 22H), 4.60 (d, J¼8.0 Hz, 1H), 3.95–4.15
(m, 6H), 0.93 (s, 9H), 0.92 (s, 9H), 0.00 (s, 3H), 0.01 (s,
3H), 0.04 (s, 3H), 0.06 (s, 3H); 13C NMR (125 MHz,
CDCl3): d¼166.3, 159.5, 144.7, 142.5, 139.0, 139.0,
1
foam. H NMR (500 MHz, CDCl3): d¼7.80 (d, J¼7.0 Hz,
2H), 7.36 (m, 2H), 6.90–7.30 (m, 18H), 6.70 (d, J¼7.0 Hz,
2H), 5.20 (dd, J¼8.0, 8.0 Hz, 1H), 5.00 (d, J¼1.8 Hz, 1H),
4.76 (d, J¼10.5 Hz, 1H), 4.70 (d, J¼2.0 Hz, 1H), 4.65 (d,
J¼12.5 Hz, 1H), 4.63 (d, J¼12.5 Hz, 1H), 4.54 (d,
J¼12.5 Hz, 1H), 3.98–4.05 (m, 2H), 3.85 (m, 2H), 3.75
(dd, J¼2.0, 12.0 Hz, 1H), 3.63 (s, 3H), 3.57 (m, 1H), 3.40
(br d, 1H), 3.20 (d, J¼12.0 Hz, 1H), 3.60 (m, 2H), 1.20 (t,
J¼7.0 Hz, 3H), 0.82 (s, 9H), 0.72 (s, 9H), 0.03 (s, 3H),
0.00 (s, 3H), ꢁ0.12 (s, 3H), ꢁ0.18 (s, 3H); LRMS calcd
for C89H112O17SSi2: 1540.72, found: 1542 (M+H).

4970
Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
ꢀ
0.35 mmol) was added drop wise and the reaction was stirred
138.9, 130.3, 128.9, 128.8, 128.7, 128.6, 128.6, 128.5, 128.4,
128.1, 128.1, 127.9, 127.8, 127.8, 127.6, 127.6, 127.3, 114.7,
102.7, 101.7, 101.2, 100.2, 99.2, 81.9, 80.1, 80.0, 74.9, 74.4,
74.2, 74.1, 73.8, 73.7, 73.3, 73.3, 72.7, 72.6, 72.2, 70.4, 69.5,
68.3, 65.8, 57.5, 55.6, 26.2, 26.0, ꢁ4.4, ꢁ4.1, ꢁ4.2, ꢁ4.8;
LRMS calcd for C133H155NO27SSi2Na: 2310, found: 2310
(M+Na).
ꢁ40 C. L-Selectride (1 M solution in THF, 0.35 mL,
ꢀ
at ꢁ40 C, then warmed to room temperature and stirred for
an additional 2 h. The reaction was quenched by adding sat-
urated aq NaHCO3 and H2O2 (33% in water, 2.4 mL). After
stirring for 30 min, the mixture was diluted with EtOAc
(100 mL) and the combined organic layers were washed
with saturated aq NaHCO3, saturated aq Na2S2O3, and brine
then dried (Na2SO4) and concentrated to afford a residue
(320 mg) of suitable purity for the next reaction. For analy-
tical data, the residue was purified over silica gel (hexane/
EtOAc¼4:1). (Rf 0.50; EtOAc/hexane¼1:4); [a]2D4 ꢁ2.6
(c¼2.05, CH2Cl2); IR (thin film) 3473, 3276, 1650, 1612,
1586, 1514, 1498, 1470, 1454, 1361, 1328, 1249, 1094,
4.1.12. Synthesis of pentasaccharide dimannosyl glycal
alcohol (37). Pentasaccharide glucal 36 (330 mg,
0.144 mmol) was azeotropically dried with benzene
(3ꢂ25 mL) then placed under high vacuum for 15 min.
Et2Oꢀ(20 mL) was added and the solution was cooled to
ꢁ40 C. Lithium aluminum hydride (1 M solution in
Et2O, 0.58 mL, ꢀ0.58 mmol) was added and the solution
was stirred at 0 C for 1 h. After diluting the solution with
saturated aq NaHCO3 (10 mL) and extracting with EtOAc
(3ꢂ15 mL), the combined organic extracts were washed
with brine (10 mL), dried (NaSO4), and concentrated in
vacuo. The residue was placed on high vacuum to afford
the crude product as a white foam (330 mg, >95%), which
was used in the next reaction without any further purifica-
tion. For analytical data, the crude product was purified
over a short column of silica gel (Rf 0.50; EtOAc/
hexane¼1:4). [a]2D4 0.9 (c¼1.73, CH2Cl2); IR (thin film)
3468, 3351, 3063, 3030, 2927, 2856, 1650, 1612, 1586,
1514, 1498, 1453, 1360, 1249, 1208, 1093, 910, 835, 735,
1
910, 836, 778 cmꢁ1; H NMR (500 MHz, CDCl3): d¼7.79
(d, J¼7.5 Hz, 2H), 7.38–7.18 (m, 54H), 6.84 (d, J¼8.5 Hz,
2H), 6.36 (d, J¼6.1 Hz, 1H), 4.96 (br s, 1H), 4.93 (br s,
1H), 4.88 (br s , 1H), 4.81 (br s, 1H), 4.90–4.39 (m, 24H),
4.33–4.30 (m, 2H), 4.28 (br s, 1H), 4.19 (br s, 1H), 4.10–
3.50 (m, 30H), 3.80 (s, 3H), 3.49–3.45 (m, 3H), 0.99 (s,
18H), 0.16 (s, 3H), 0.12 (s, 6H), 0.11 (s, 3H); 13C NMR
(125 MHz, CDCl3): d¼159.1, 144.3, 141.7, 138.8, 138.7,
138.6, 138.5, 138.2, 138.1, 138.1, 138.0, 137.7, 137.6,
132.1, 130.1, 128.7, 128.5, 128.4, 128.3, 128.2, 128.2,
128.1, 128.1, 128.0, 128.0, 128.0, 127.9, 127.6, 127.4,
127.4, 127.3, 127.3, 127.2, 127.1, 113.7, 102.2, 101.1,
100.6, 100.5, 100.1, 84.2, 80.1, 79.7, 75.9, 75.7, 75.3,
74.9, 74.5, 74.5, 73.4, 73.4, 73.1, 73.1, 73.0, 72.9, 72.8,
72.3, 70.3, 70.2, 69.8, 69.2, 69.0, 67.6, 66.5, 58.4, 55.1,
25.8, 18.1, ꢁ4.6, ꢁ4.7, ꢁ4.8, ꢁ5.0; FAB(+)MS: 2207,
2132, 2115, 1990; HRMS calcd for C126H151NO26SSi2Na:
2204.9680, found: 2204.9700.
698 cmꢁ1
;
1H NMR (500 MHz, CDCl3): d¼7.75 (d,
J¼7.5 Hz, 2H), 7.45–7.18 (m, 45H), 6.81 (d, J¼8.6 Hz,
2H), 6.31 (d, J¼6.1 Hz, 1H), 4.93–4.84 (m, 6H), 4.70–
4.41 (m, 24H), 4.29 (d, J¼7.6 Hz, 1H), 4.14–4.01 (m, 7H),
3.98–3.71 (m, 14H), 3.79 (s, 3H), 3.67–3.62 (m, 2H),
3.53–3.36 (m, 9H), 3.28–3.19 (m, 2H), 0.96 (s, 9H), 0.92
(s, 9H), 0.12 (s, 3H), 0.06 (s, 3H), 0.03 (s, 3H), 0.02 (s,
3H); 13C NMR (125 MHz, CDCl3): d¼159.1, 144.3, 141.7,
138.9, 138.6, 138.6, 138.5, 138.3, 138.2, 138.1, 137.5,
132.1, 130.0, 128.6, 128.5, 128.4, 128.4, 128.3, 128.2,
128.2, 128.1, 128.1, 128.1, 128.0, 127.9, 127.7, 127.6,
127.5, 127.4, 127.4, 127.4, 127.3, 127.3, 127.2, 127.1,
113.8, 103.0, 102.3, 101.1, 100.7, 100.3, 80.0, 79.1, 79.0,
76.8, 75.8, 75.0, 75.0, 74.8, 74.5, 74.5, 74.0, 73.8, 73.4,
73.1, 73.0, 72.9, 72.5, 72.0, 71.8, 70.4, 70.0, 69.2, 69.1,
69.0, 68.7, 67.5, 66.0, 58.7, 55.2, 25.7, 25.7, 18.0, ꢁ4.6,
ꢁ4.7, ꢁ4.8, ꢁ5.0; FAB(+)MS: 2205, 2192, 2130, 2115,
2110, 1988; HRMS calcd for C126H151NO26SSi2Na:
2204.9681, found: 2204.9680.
4.1.14. Synthesis of acetylated pentasaccharide triman-
nosyl glycal alcohol 39. The crude alcohol (320 mg) was
dissolved in CH2Cl2 (8 mL) then treated with Ac2O
(1.2 mL, 12.7 mmol), Et3N (1.2 mL, 14.3 mmol), and
DMAP (5 mg, 0.04 mmol) at room temperature for 15 h.
The reaction mixture was diluted with EtOAc (100 mL),
and the organic layer was washed with saturated aq NaHCO3
(15 mL) and brine (15 mL) then dried (Na2SO4) and concen-
trated under reduced pressure. The residue was purified over
silica gel to afford 39 (270 mg, 0.119 mmol, 82% in four
1
steps) as a white foam. Rf 0.5 (hexane/EtOAc¼1:1); H
NMR (500 MHz, CDCl3): d¼7.98 (d, J 7.5 Hz, 2H), 7.20–
7.50 (m, 57H), 6.74 (d, J¼6.5 Hz, 2H), 6.35 (d, J¼6.5 Hz,
1H), 5.44 (s, 1H), 5.40 (d, J¼8.0 Hz, 1H), 4.40–5.00 (m,
36H), 3.60–3.80 (m, 22H), 3.58 (m, 4H), 2.08 (s, 3H), 1.97
(s, 3H), 0.89 (s, 9H), 0.86 (s, 9H), 0.02 (s, 3H), 0.01 (s,
3H), 0.00 (s, 3H), ꢁ0.02 (s, 3H); LRMS calcd for
C130H155NO28SSi2Na: 2289, found: 2289 (M+Na).
4.1.13. Synthesis of pentasaccharide trimannosyl glycal
alcohol 38. Glycal alcohol 37 (330 mg, 0.144 mmol) was
azeotropically dried with toluene (3ꢂ5 mL) then placed un-
der high vacuum for 15 h. The residue was dissolved in
CH2Cl2 (9 mL) and pyridine (106 mL, 1.23 mmol) was
added. In an argon-filled glove bag, Dess–Martin reagent
(330 mg, 0.77 mmol) was added in one portion. The result-
ing mixture was stirred at room temperature until the starting
material had been consumed (about 1 h). At that point, the
reaction was quenched by adding saturated aq Na2S2O3
(1 mL) and diluted with EtOAc (100 mL). The organic layer
was washed with saturated aq Na2S2O3 (10 mL) and brine
(10 mL) then dried (Na2SO4) and concentrated to afford
the intermediate keto-compound (330 mg) pure enough for
further use. After azeotropically drying the residue with tol-
uene (3ꢂ), it was dissolved in dry THF (8 mL) and cooled to
4.1.15. Synthesis of ethylthio pentasaccharide 41. Penta-
saccharide glycal 39 (353 mg, 0.155 mmol) and benzenesul-
fonamide (98 mg, 0.62 mmol) were combined and
azeotropically dried with benzene (3ꢂ5 mL). The residue
was dissolved in CH2Cl2 (15 mL), mixed with freshly
˚
dried 4 A MS (1.5 g), and stirred at room temperature for
30 min. Iodonium-di-sym-collidine perchlorate (583 mg,
1.25 mmol) in CH2Cl2 (5 mL) was added via cannula to
the mixture, and the resulting suspension was stirred at
ꢀ
0 C for 30 min at which point it was warmed to room tem-
perature and filtered through a short pad of Celite. The solids

Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
4971
were washed with EtOAc and the combined filtrates were
washed with saturated Na2S2O3 (ꢂ2), CuSO4 (ꢂ4), brine,
Na2S2O3, and brine. The organic layer was dried (Na2SO4)
and concentrated in vacuo. Purification over silica gel (hex-
ane/EtOAc¼2:1) yielded the intermediate iodosulfonamide,
which was taken on crude. The iodosulfonamide was subse-
DMAP (10 mg, 0.08 mmol). The resulting mixture was
stirred at room temperature for 15 h then concentrated at re-
duced pressure. The residue was dissolved in EtOAc
(100 mL), washed with saturated aq NaHCO3 and brine
then dried (Na2SO4) and concentrated. Purification over
silica gel (EtOAc/MeOH¼95:5) yielded the peracetylated
thioglycoside 45 (31 mg, 0.020 mmol, 77%) as a slightly
yellow foam. Rf 0.5 (EtOAc/MeOH¼95:5); [a]2D4 ꢁ15
(c¼2.2, CHCl3); IR (thin film): 2900, 1750, 1420, 1280,
ꢀ
quently dissolved in DMF (5 mL) and cooled to ꢁ40 C. A
solution of LiHMDS (1 M in THF, 467 mL, 0.47 mmoꢀ l) and
ethanethiol (80 mL, 1.08 mmol) was cooled to ꢁ40 C and
added via cannula to the solution containing the iodosulfo-
1050 cmꢁ1
;
1H NMR (500 MHz, CDCl3): d¼6.11 (d,
ꢀ
namide. The mixture was stirred at ꢁ40 C for 3 h and sub-
J¼9.3Hz, 1H), 5.59 (d, J¼9.7 Hz, 1H), 5.58–5.20 (m, 6H),
5.20–4.90 (m, 8H), 4.76 (s, 1H), 4.57 (s, 1H), 4.42–3.90
(m, 19H), 2.62 (m, 2H), 2.59–1.76 (16s, 48H), 1.20 (t,
J¼7.3 Hz, 3H); 13C NMR (500 MHz, CDCl3): d¼171.0,
170.8, 170.7, 170.6, 170.5, 170.4, 170.3, 170.2, 170.1,
169.9, 169.8, 169.7, 113.7, 101.2, 98.6, 97.1, 96.7, 84.4,
77.3, 77.0, 76.8, 76.0, 74.1, 72.7, 72.5, 72.0, 69.7, 69.3,
69.2, 69.2, 68.8, 68.3, 68.2, 66.8, 65.6, 65.5, 62.4, 62.2,
62.1, 60.4, 54.2, 52.9, 24.3, 23.2, 23.0, 21.0, 20.9, 20.9,
20.8, 20.8, 20.7, 20.6, 20.6, 19.7, 14.7, 14.2; HRMS calcd
for C64H90N2O39S: 1542.4840, found: 1542.4863.
sequently diluted with ether (150 mL), washed with
saturated aq NaHCO3 and brine then dried (Na2SO4) and
concentrated. The residue was purified over silica gel (hex-
ane/EtOAc¼2:1) to afford the desired thioglycoside 41
(293 mg, 0.12 mmol, 81%) as white foam. [a]2D4 10
(c¼1.3, CHCl3); IR (thin film): 3275, 2925, 2855, 1747,
1
1453, 1361, 1328, 1247, 1157, 1092, 736 cmꢁ1; H NMR
(500 MHz, CDCl3): d¼7.00–8.00 (m, 64H), 6.75 (d,
J¼6.4 Hz, 2H), 5.60 (d, J¼9.0 Hz, 1H), 5.35 (d, J¼2.8 Hz,
1H), 5.15 (s, 2H), 4.90 (s, 1H), 4.40–4.85 (m, 33H), 4.20
(d, J¼7.1 Hz, 1H), 3.72 (s, 3H), 3.40–3.80 (m, 27H), 2.50
(m, 2H), 2.10 (s, 3H), 1.10 (t, J¼7.6 Hz, 3H), 0.90 (s, 9H),
0.88 (s, 9H), 0.02 (s, 6H), 0.01 (s, 6H), 0.00 (s, 6H);
HRMS calcd for C138H166N2O30S3Si2: 2483.0225, found:
2483.0264 (M+H).
4.1.18. Synthesis of deacetylated thioglycoside 46. To a so-
lution of the peracetylated pentasaccharide 45 (30 mg,
0.02 mmol) in MeOH (2 mL) was added NaOMe (25% solu-
tion in MeOH, 30 mL, 0.13 mmol). The mixture was stirred
at room temperature for 15 h then purified directly using li-
pophilic LH-20 Sephadex. The appropriate fractions were
combined and concentrated in a lyophilizer to afford the thio-
4.1.16. Synthesis of the desilylated pentasaccharide thio-
glycoside 43. The thioglycoside 41 (170 mg, 0.07 mmol) in
THF (5 mL) was stirred with TBAF (1 M solution in THF,
0.7 mL, 0.70 mmol) at room temperature for 36 h. At that
point, the solution was concentrated and the residue was
diluted with EtOAc. The organic layer was washed with
saturated aq NH4Cl and brine then dried (Na2SO4) and con-
centrated. The residue was purified over silica gel (hexane/
EtOAc¼1:1, then 2:3) to afford the desired diol 43
(120 mg, 0.054 mmol, 80%) as a white foam. Rf 0.25
glycoside 46 (16 mg, 16.7 mmol, 86%). Rf 0.80 (EtOAc/
1
iPrOH/H2O¼1:1:1); H NMR (500 MHz, D2O, 24 C):
ꢀ
d¼5.12 (s, 1H), 4.93 (s, 1H), 4.68 (d, J¼10.7 Hz, 1H),
4.62 (d, J¼8.9 Hz, 1H), 4.27 (s, 1H), 4.08 (s, 1H), 3.99 (s,
1H), 3.52–3.96 (m, 30H), 2.75 (m, 2H), 2.10 (s, 3H), 2.05
(s, 3H), 1.25 (t, J¼7.4 Hz, 3H); 13C NMR (125 MHz,
ꢀ
D2O, 24 C): d¼174.8, 173.6, 104.4, 103.3, 102.2, 101.3,
85.9, 82.8, 81.3, 80.8, 76.8, 75.8, 75.4, 74.7, 73.9, 72.7,
72.2, 71.9, 69.4, 69.1, 68.1, 63.4, 63.2, 62.0, 61.6, 56.7,
55.8, 50.1, 25.2, 23.5, 23.1, 15.4; HRMS calcd for
C36H62N2O25S: 954.3362, found: 954.3382.
1
(hexane/EtOAc¼1:1); [a]2D4 ꢁ6 (c¼1.5, CHCl3); H NMR
(500 MHz, CDCl3): d¼7.86 (d, J¼7.4 Hz, 2H), 7.76 (d,
J¼7.4 Hz, 2H), 7.00–7.40 (m, 60H), 6.75 (d, J¼8.5 Hz,
2H), 5.70 (d, J¼6.4 Hz, 1H), 5.25 (s, 1H), 5.10 (s, 1H),
4.90 (s, 1H), 4.30–4.60 (m, 33H), 4.10–4.20 (m, 2H),
3.40–4.00 (m, 27H), 3.35 (m, 2H), 3.20 (m, 2H), 2.95 (d,
J¼7.0 Hz, 2H), 2.4 (m, 2H), 2.08 (s, 3H), 1.10 (t,
J¼6.4 Hz, 3H); 13C NMR (125 MHz, CDCl3): d¼169.2,
158.3, 140.5, 137.4, 137.2, 137.1, 136.9, 136.8, 128.2,
127.5, 127.4, 127.3, 127.3, 127.2, 127.1, 127.0, 126.9,
126.8, 126.8, 126.8, 126.7, 126.6, 126.3, 126.9, 100.7,
98.9, 98.5, 97.0, 93.0, 86.6, 81.7, 79.3, 78.6, 78.6, 78.0,
77.7, 72.4, 72.3, 72.3, 71.0, 70.6, 70.4, 67.8, 67.6, 67.0,
43.8, 37.8, 23.8, 18.8, 13.7; LRMS calcd for
C124H136N2O29S3: 2213, found: 2236 (M+Na) positive,
2248 (M+Cl) negative.
4.1.19. Synthesis of free pentasaccharide 47. To a solution
of ethylthioglycoside 46 (16 mg, 16.7 mmol) in H2O (5 mL)
was added HgCl2 (30 mg, 83.3 mmol) and then CaCO3
(30 mg, 167 mmol). The mixture was stirred for 15 h at
room temperature and then purified via size-exclusion chro-
matography. The appropriate fractions were collected and
lyophilized to afford 47 (16 mg, 17.5 mmol, 95%) as a a/b
mixture of anomers (2:3 or 3:2). Rf 0.70 (EtOAc/iPrOH/
1
ꢀ
H2O¼1:1:1); H NMR (500 MHz, D2O, 24 C): d¼5.20
(s, 1H), 5.11 (s, 1H), 4.99 (s, 1H), 4.26 (s, 2H), 4.08–3.43
(m, 30H), 2.08 (s, 3H), 2.04 (s, 3H); LRMS calcd for
C34H58N2O26: 910, found: 933 (M+Na), 915 (MꢁH2O+Na).
4.1.17. Synthesis of peracetylated thioglycoside pentasac-
charide 45. To a deep blue solution of sodium metal (58 mg,
4.1.20. Synthesis of glycosylamine 11. To the solution of
free sugar 47 (16 mg, 0.018 mmol) in H2O (14 mL) was
added solid NH4HCO3 (9.0 g, 114 mmol). The resulting
mixture was stirred at room temperature until all the starting
material was completely converted (monitored by 1H NMR
and TLC, EtOAc/iPrOH/H2O¼1:1:1). During this period,
NH4HCO3 needs to be continually added to keep the solu-
tion saturated. After completion of the reaction, the stirring
bar was removed and the solution was lyophilized. The solid
ꢀ
2.52 mmol) in NH3 (w10 mL) at ꢁ78 C was added a solu-
tion of thioglycoside 43 (58 mg, 0.03 mmol) in THF (2 mL).
ꢀ
The solution was stirred at ꢁ78 C for 45 min then
quenched by the addition of solid NH4Cl (96 mg,
1.79 mmol) and MeOH (1 mL). The volatiles were removed
under a stream of N2 and the residue was treated with Ac2O
(2 mL, 21.2 mmol), pyridine (2 mL, 24.7 mmol), and

4972
Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
material was several times redissolved in H2O (5 mL) and
lyophilized until the residue exhibited a constant weight. A
white powder (17 mg, 18.7 mmol, >99%) was obtained. Rf
0.34 (EtOAc/iPrOH/H2O¼1:1:1); 1H NMR (500 MHz,
and concentrated in vacuo. The residue was purified by silica
gel chromatography (hexane/EtOAc¼10:1) to afford 58
(13.58 g, 17 mmol, 91%) as a white foam. [a]2D5 ꢁ9.8
(c¼3.9, CHCl3); IR (thin film): 3028, 2865, 1650, 1453,
1097 cmꢁ1
;
1H NMR (500 MHz, CDCl3): d¼7.71–7.24
ꢀ
D2O, 24 C): d¼5.12 (s, 1H), 4.93 (d, J¼1.4 Hz, 1H), 4.62
(d, J¼7.8 Hz, 1H), 4.27 (s, 1H), 4.16 (d, J¼9.0 Hz, 1H),
4.08 (m, 1H), 3.98 (m, 3H), 3.48–3.94 (br m, 28H), 2.09
(s, 3H), 2.05 (s, 3H); HRMS calcd for C34H59N3O25Na:
932.3437, found: 932.3468 (M+Na).
(m, 25H), 6.45 (d, J¼6.2 Hz, 1H), 5.93 (m, 1H), 5.28 (dd,
J¼1.6, 12.3 Hz, 1H), 5.18 (dd, J¼1.5, 10.4 Hz, 1H), 4.97
(d, J¼12.3 Hz, 1H), 4.87 (m, 1H), 4.74 (m, 2H), 4.66–4.44
(m, 7H), 4.38 (m, 2H), 4.26 (m, 1H), 4.17 (m, 4H), 3.85–
3.31 (m, 8H); 13C NMR (125 MHz, CDCl3): d¼144.4,
138.8, 138.7, 138.1, 137.9, 128.4, 128.3, 128.2, 128.2,
128.1, 127.9, 127.7, 127.6, 127.5, 127.4, 127.3, 82.0, 79.3,
75.9, 75.1, 73.6, 73.5, 73.3, 73.2, 72.3, 71.8, 70.3, 68.6,
68.0; HRMS (FAB) calcd for C50H54O9Na: 821.3666,
found: 821.3666.
4.1.21. Synthesis of the N-linked pentasaccharide–penta-
peptide adduct 1. Pentasaccharide glycosylamine 11 (4 mg,
4.40 mmol) and L-pentapeptide 4837 (3.68 mg, 6.60 mmol)
were mixed in anhydrous DMSO (0.5 mL) and HOBt
(2.97 mg, 22 mmol), HBTU (9.48 mg, 22 mmol), and
Hunig’s base (15 mL, 8.8 mmol) were added. After stirring
¨
for 2 d at room temperature, the reaction mixture was lyoph-
ilized and purified on a reverse-phase Vydac C18-column
(4.6ꢂ250 mm) to afford 1 (2.5 mg, 1.72 mmol, 40%).
HPLC gradient: A: 0.1% TFA/H2O, B: 0.09% TFA/70%
CH3CN/H2O, gradient 5/50% B over 23 min; 50/
100% B over 5 min; glycopeptide elutes atꢀ21 min. 1H
NMR (800 MHz, H2O, Watergate method, 24 C): 8.52 (d,
J¼8.9 Hz, 1H), 8.33 (d, J¼9.6 Hz, 1H), 8.33 (d, J¼7.3 Hz,
1H), 8.22 (d, J¼5.9 Hz, 1H), 8.12 (d, J¼8.2 Hz, 2H), 8.08
(d, J¼8.0 Hz, 1H), 7.92 (d, J¼8.0ꢀHz, 1H), 7.42 (s, 1H),
7.04 (s, 1H); (500 MHz, D2O, 30 C) d¼5.24 (s, 1H, H-
1c), 5.15 (d, J¼9.7 Hz, 1H, H-1a), 5.04 (s, 1H, H-1d), 4.90
(s, 1H, H-1d), 4.84 (t, J¼6.3 Hz, 1H), 4.76 (d, J¼12.6 Hz,
1H, H-1b), 4.33–4.48 (m, 22H), 4.19 (s, 1H), 3.70–4.10
(m, 18H), 3.00 (dd, J¼6.4, 16.4 Hz, 1H), 2.87 (dd, J¼6.4,
16.7 Hz, 1H), 2.30 (m, 1H), 2.20 (s, 3H, Ac), 2.15 (s, 3H,
Ac), 2.13 (s, 3H, Ac), 1.76 (m, 1H), 1.50 (d, J¼7.2 Hz,
3H, CH3), 1.34 (d, J¼6.4 Hz, 3H, CH3), 1.07 (d,
J¼6.5 Hz, 6H, 2CH3), 1.02 (d, J¼6.4 Hz, 3H, CH3); 13C
4.1.24. Synthesis of iodosulfonamide. To a flask containing
lactal 58 (6.0 g, 7.51 mmol), 2-(trimethylsilyl)ethanesulfo-
˚
namide (5.44 g, 30.0 mmol), and powdered 4 A molecular
sieves (3.0 g, freshly flame-dried) was added CH2Cl2
(30 mL) and the mixture was stirred at room tempera-
ꢀ
ture for 30 min. The mixture was cooled to 0 C and I(sym-
collidine)2ClO4 (2.23 g, 4.76 mmol), prepared in situ from
Ag(collidine)2ClO4 (10.13 g, 22.53 mmol) and 5.52 g I2
˚
(21.78 mmol) in the presence of powdered 4 A molecular
sieves (3.0 g, freshly flame-dried) in CH2Cl2 (130 mL) was
added via cannula. The reaction was stirred for 1.5 h, then
diluted with CH2Cl2 (150 mL) and filtered. The filtrate
was washed with saturated aq Na2S2O3 (3ꢂ75 mL), satu-
rated aq CuSO4 (5ꢂ75 mL), saturated aq Na2S2O3
(1ꢂ75 mL), and brine (1ꢂ75 mL). The organic layer was
dried over Na2SO4 and concentrated in vacuo. The residue
was purified by silica gel chromatography (hexane/
EtOAc¼4:1 to 3:1) to afford the desired diaxial isomer
(6.26 g, 5.65 mmol, 75%) as well as a small amount of the
diequatorial isomer (0.589 g, 0.90 mmol, 12%). [a]D25
ꢁ12.6 (c¼0.9, CHCl3); IR (thin film): 3258, 3029, 2867,
ꢀ
(200 MHz, H2O, Watergate method, 24 C): d¼105.2
(C-1), 104.2 (C-1), 103.3 (C-1), 102.5 (C-1), 81 (C-1a);
HRMS (FAB) calcd for C58H99N9O33Na: 1472.6237, found:
1472.6282.
1455, 1337, 1107 cmꢁ1 1H NMR (500 MHz, CDCl3):
;
d¼7.46–7.22 (m, 25H), 5.93 (m, 1H), 5.34 (dd, J¼1.6,
17.2 Hz, 1H), 5.20 (m, 2H), 4.93–4.37 (m, 12H), 4.19 (m,
4H), 4.08 (m, 1H), 3.85–3.34 (m, 10H), 3.04 (m, 1H), 1.06
(m, 2H), 0.07 (s, 9H); 13C NMR (125 MHz, CDCl3):
d¼138.5, 138.3, 137.9, 137.7, 137.1, 128.5, 128.4, 128.3,
128.2, 128.1, 128.0, 128.0, 127.9, 127.9, 127.8, 127.8,
127.7, 127.7, 127.6, 127.5, 127.4, 116.8, 103.7, 81.8, 79.6,
79.1, 79.0, 78.2, 75.3, 74.6, 74.4, 73.9, 73.6, 73.5, 73.4,
73.1, 72.4, 71.9, 68.9, 67.4, 51.0, 29.8, 10.4, ꢁ2.1; HRMS
(FAB) calcd for C55H68INO11SSi: 1105.3286, found:
1105.3290.
4.1.22. Synthesis of the N-linked pentasaccharide–penta-
peptide adduct 2. Same procedure as in the synthesis of 1.
1
ꢀ
H NMR (500 MHz, D2O, 36 C): 5.01 (s, 1H), 4.93 (d,
J¼9.5 Hz, 1H), 4.81 (s, 1H), 4.67 (s, 1H), 4.60 (t,
J¼7.0 Hz, 1H), 4.52 (d, J¼7.5 Hz, 1H), 4.18–4.25 (m,
2H), 4.19 (m, 2H), 3.98 (s, 1H), 3.88 (s, 1H), 2.75 (dd,
J¼6.5, 15.0 Hz, 1H), 2.60 (m, 2H), 2.10 (m, 1H), 1.97 (s,
3H), 1.93 (s, 3H), 1.90 (s, 3H), 1.78 (br s, 1H), 1.58 (m,
1H), 1.50 (m, 2H), 1.28 (d, J¼7.0 Hz, 3H), 1.12 (d,
J¼7.0 Hz, 3H), 0.87 (s, 6H), 0.81 (d, J¼6.4 Hz, 3H).
4.1.25. Synthesis of sulfonamidoethylthioglycoside 59.
The iodosulfonamide (6.26 g, 5.65 mmol) was dissolved in
DMF (20 mL) and added drop wise to a solution of ethane-
thiol (2.09 mL, 28.25 mmol, 5 equiv) and LiHMDS (1 M so-
lution in THF, 17 mL, 17 mmol) in DMF (80 mL) and
4.1.23. Synthesis of allyl lactal 58. 30-Allyl lactal38 (6.50 g,
18.68 mmol) was suspended in DMF (160 mL) and cooled
ꢀ
to 0 C. NaH (60% in oil, 5.98 g, 149.44 mmol) wasꢀadded
in one portion and the reaction was maintained at 0 C for
30 min. Benzyl bromide (22.2 mL, 186.8 mmol) was added
drop wise and the reaction was stirred at room temperature
ꢀ
cooled to ꢁ40 C. The reaction was allowed to slowly
warm to room temperature and stirred for 12 h. Most of
the solvent (DMF) was removed by evaporation under
high vacuum and the residue was diluted with saturated aq
NaHCO3 (100 mL) and washed with EtOAc (3ꢂ100 mL).
The organic layer was dried over Na2SO4 and concentrated
in vacuo. The residue was purified by silica gel chromato-
graphy (hexane/EtOAc¼3.5:1) to afford 59 (5.0 g,
ꢀ
for 24 h. The reaction was cooled to 0 C and quenched
by drop wise addition of glacial AcOH (2.24 g, 37 mmol).
Most of the solvent (DMF) was removed by evaporation
under high vacuum and the residue was diluted with satu-
rated aq NaHCO3 (100 mL) and washed with EtOAc
(5ꢂ100 mL). The organic layer was dried over Na2SO4

Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
4973
4.80 mmol, 85%) as a white glass. [a]2D5 ꢁ10.2 (c¼3.1,
128.2, 128.2, 128.1, 128.0, 127.8, 127.7, 127.7, 127.6,
127.5, 127.4, 127.3, 127.2, 126.8, 123.4, 123.2, 116.4,
102.9, 82.2, 80.9, 80.0, 79.7, 78.1, 77.8, 75.4, 74.5, 74.3,
73.6, 73.4, 73.0, 71.5, 68.3, 68.2, 54.8, 23.7, 14.9; HRMS
(FAB) calcd for C60H63NO11SNa: 1028.4019, found:
1028.4040.
CHCl3); IR (thin film): 3268, 3029, 2868, 1454,
;
1091 cmꢁ1
1H NMR (500 MHz, CDCl3): d¼7.38–7.18
(m, 25H), 5.92 (m, 1H), 5.34 (d, J¼17.5 Hz, 1H), 5.19 (d,
J¼10.5 Hz, 1H), 4.93 (d, J¼11.8 Hz, 2H), 4.77–4.36 (m,
12H), 4.16 (m, 2H), 3.97–3.37 (m, 13H), 3.13 (m, 2H),
2.71 (m, 2H), 1.28 (t, J¼7.4 Hz, 3H), 1.05 (m, 2H), ꢁ0.01
(s, 9H); 13C NMR (125 MHz, CDCl3): d¼138.7, 138.6,
138.2, 137.9, 134.8, 128.3, 128.3, 128.2, 128.1, 128.1,
128.0, 127.9, 127.9, 127.8, 127.8, 127.7, 127.7, 127.7,
127.6, 127.6, 127.5, 127.5, 127.4, 127.4, 127.3, 116.3,
103.1, 83.8, 82.0, 81.2, 79.8, 79.7, 76.6, 75.2, 74.5, 73.8,
73.4, 73.2, 73.0, 71.6, 68.3, 67.9, 58.0, 24.0, 14.8, ꢁ2.0.
4.1.28. Synthesis of 30-hydroxy ethylthioglycoside. The
phthalamidoethylthioglycoside (4.25 g, 4.22 mmol) was
dissolved in EtOH/H2O (80 mL, 1:1 v/v). RhCl(PPh3)2
(0.39 g, 0.42 mmol) and DABCO (0.947 g, 8.44 mmol)
ꢀ
were added and the reaction was heated to 90 C for 3 h.
At that point, the reaction was cooled to room temperature
and diluted with THF (50 mL) and 1 N HCl (40 mL). The
mixture was stirred at room temperature for 48 h. The or-
ganic solvents and water were removed by evaporation and
the residue was dissolved in EtOAc (150 mL) then washed
with saturated aq NaHCO3 (3ꢂ50 mL), dried over
Na2SO4, and concentrated in vacuo. The residue was purified
by silica gel chromatography (hexane/EtOAc¼5:1) to afford
the title compound (3.0 g, 3.1 mmol, 74%) as a white foam.
[a]2D5 22.1 (c¼0.6, CHCl3); IR (thin film): 3472, 3029, 2868,
1774, 1713, 1307, 1078 cmꢁ1; 1H NMR (500 MHz, CDCl3):
d¼7.79 (m, 1H), 7.67 (m, 3H), 7.36–7.22 (m, 20H), 6.98 (m,
2H), 6.86 (m, 3H), 5.23 (d, J¼10.1 Hz, 1H), 4.87 (m, 2H),
4.73 (m, 2H), 4.58 (m, 2H), 4.47–4.30 (m, 7H), 4.07 (m,
1H), 3.82 (m, 2H), 3.51 (m, 6H), 2.68 (m, 2H), 1.15 (t,
J¼7.4 Hz, 3H); 13C NMR (125 MHz, CDCl3): d¼168.1,
167.5, 138.8, 138.7, 138.4, 138.3, 137.9, 131.7, 128.5,
128.4, 128.3, 128.2, 127.9, 127.8, 127.8, 127.7, 127.7,
127.6, 127.5, 127.5, 127.4, 126.9, 123.5, 102.9, 80.9, 80.7,
79.6, 78.0, 77.9, 75.9, 74.9, 74.4, 74.0, 73.4, 73.3, 73.1,
68.2, 68.1, 54.7, 23.8, 14.9; HRMS (FAB) calcd for
C57H59NO11SNa: 988.3706, found: 988.3744.
4.1.26. Synthesis of ethylthioglycoside lactosamine. To the
solution of thiosulfonamide 59 (5.00 g, 4.80 mmol) in DMF
(500 mL) was added powderedꢀCsF (3.64 g, 24.0 mmol) and
the mixture was stirred at 90 C for 2 d. The solution was
cooled to room temperature and most of the solvent
(DMF) was removed by evaporation under high vacuum.
The remaining residue was diluted with saturated aq
NaHCO3 (100 mL) and washed with Et2O (3ꢂ100 mL).
The organic layer was dried over Na2SO4 and concentrated
in vacuo. The residue was purified by silica gel chromatog-
raphy (hexane/EtOAc¼1:1 to 1:2 with 2% of Et3N v/v) to af-
ford the title compound (3.96 g, 3.75 mmol, 94%) as a white
glass. [a]2D5 ꢁ12.2 (c¼1.0, CHCl3); IR (thin film): 3029,
2921, 2867, 1453, 1090 cmꢁ1
;
1H NMR (500 MHz,
CDCl3): d¼7.48–7.22 (m, 25H), 5.92 (m, 1H), 5.34 (dd,
J¼1.7, 7.2 Hz, 1H), 5.19 (m, 2H), 4.97 (d, J¼11.1 Hz,
1H), 4.81 (m, 2H), 4.57–4.28 (m, 8H), 4.18 (d, J¼5.3 Hz,
2H), 3.99–3.17 (m, 5H), 3.53–3.33 (m, 6H), 2.95 (m, 1H),
2.72 (m, 2H), 1.29 (t, J¼7.5 Hz, 3H); 13C NMR
(125 MHz, CDCl3): d¼139.0, 138.9, 138.8, 138.0, 134.9,
128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7,
127.6, 127.5, 127.4, 127.3, 119.4, 102.7, 86.5, 84.8, 82.3,
79.9, 79.8, 76.2, 75.3, 75.2, 74.6, 73.4, 73.0, 71.6, 68.4,
68.1, 55.7, 24.1, 15.4; HRMS (FAB) calcd for
C52H61NO9S: 876.4145, found: 876.4156.
Propyl phthalamidoethylthioglycoside (product of allylic
double bond reduction) was isolated as a by-product
(0.62 g, 15%). If DABCO is excluded, the yield of desired
product is much higher (91%), but isomerization required
3 d to reach completion.
4.1.27. Synthesis of phthalamidoethylthioglycoside. To
a solution of the lactosamine (3.70 g, 4.22 mmol) in pyridine
(60 mL) was added phthalic anhydride (0.94 g, 6.33 mmol)
and the reaction was stirred at room temperature for
45 min. Pivalic anhydride (4.72 g, ꢀ25.32 mmol) was added
and the reaction was stirred at 85 C for 5 d. The solution
was cooled to room temperature and the pyridine was re-
moved by evaporation. The remaining residue was dissolved
in EtOAc (150 mL) and washed with saturated aq NaHCO3
(3ꢂ50 mL). The organic layer was dried over Na2SO4 and
concentrated in vacuo. The residue was purified by silica
gel chromatography (hexane/EtOAc¼10:1) to afford the ti-
tle compound (4.25 g, 4.22 mmol, >99%) as a white foam.
[a]2D5 22.2 (c¼0.7, CHCl3); IR (thin film): 3029, 3026,
4.1.29. Synthesis of ethylthioglycoside donor 60. To a solu-
tion of 30-hydroxy ethylthioglycoside (3.0 g, 3.1 mmol) in
CH2Cl2 (10 mL) was added chloroacetic anhydride (2.65 g
15.5 mmol), DMAP (cat.), and di-tert-butylpyridine
(DTBP, 1.78 g, 9.3 mmol) and the reaction was stirred at
room temperature for 24 h. The solution was diluted with
CH2Cl2 (100 mL) and washed with saturated aq NaHCO3
(3ꢂ50 mL) and brine. The organic layer was dried over
Na2SO4 and concentrated in vacuo. The residue was purified
by silica gel chromatography (hexane/EtOAc¼10:1) to af-
ford 60 (3.142 g, 3.02 mmol, 97%) as a white foam. [a]D25
49.5 (c¼0.2, CHCl3); IR (thin film): 2922, 1771, 1713,
1
1386, 1076 cmꢁ1; H NMR (500 MHz, CDCl3): d¼7.83
2923, 2867, 1774, 1713, 1386, 1086 cmꢁ1
;
1H NMR
(m, 1H), 7.71 (m, 3H), 7.37–7.22 (m, 20H), 6.98 (m, 2H),
6.87 (m, 3H), 5.23 (d, J¼10.1 Hz, 1H), 4.84 (m, 3H),
4.65–4.26 (m, 12H), 4.10 (m, 1H), 3.93 (d, J¼3.2 Hz, 1H),
3.87 (m, 1H), 3.77–3.42 (m, 7H), 2.69 (m, 2H), 1.19 (t,
J¼7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3): d¼168.1,
167.5, 166.6, 138.3, 138.2, 138.1, 137.9, 137.8, 133.8,
133.7, 131.6, 128.5, 128.4, 128.3, 128.3, 128.2, 128.0,
127.9, 127.8, 127.7, 127.7, 127.7, 127.6. 127.5, 127.4,
126.9, 123.5, 123.2, 102.7, 80.9, 79.5, 77.9, 77.8, 77.6,
(500 MHz, CDCl3): d¼7.81 (m, 1H), 7.67 (m, 3H), 7.37–
7.21 (m, 20H), 6.97 (m, 2H), 6.85 (m, 3H), 5.94 (m, 1H),
5.35 (dd, J¼1.7, 7.2 Hz, 1H), 5.25 (d, J¼10.0 Hz, 1H),
5.19 (dd, J¼1.1, 10.5 Hz, 1H), 4.92–4.81 (m, 4H), 4.56–
4.17 (m, 10H), 4.16 (m, 2H), 3.86 (m, 1H), 3.76 (m, 2H),
3.47–3.32 (m, 7H), 2.68 (m, 2H), 1.18 (t, J¼8.4 Hz, 3H);
13C NMR (125 MHz, CDCl3): d¼168.1, 167.5, 139.1,
138.8, 138.5, 138.1, 134.9, 133.8, 133.7, 131.7, 128.4,

4974
Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
75.1, 74.9, 74.7, 74.5, 73.3, 73.1, 72.6, 67.9, 67.4, 54.6, 40.5,
23.7, 14.9; HRMS (FAB) calcd for C59H60ClNO12SNa:
1064.3422, found: 1064.3386.
9H); 13C NMR (125 MHz, CDCl3): d¼138.7, 138.1, 138.0,
137.5, 137.1, 128.5, 128.4, 128.4, 128.3, 128.3, 128.2,
128.1, 128.1, 128.0, 127.9, 127.8, 127.7, 127.4, 127.3,
126.4, 102.9, 97.3, 84.2, 80.0, 79.6, 78.2, 77.9, 77.9, 75.4,
75.0, 74.8, 74.6, 74.4, 74.0, 73.7, 73.6, 73.5, 73.0, 72.7,
72.1, 72.0, 71.6, 69.3, 67.5, 66.2, 51.0, 30.5, 17.2, 10.4,
ꢁ1.9; HRMS (FAB) calcd for C79H92INO15SSi:
1504.4899, found: 1504.4963.
4.1.30. Synthesis of perbenzylated H-type 2 trisaccha-
ride. To a solution of glycal 6339 (9.9 g, 9.07 mmol) in
THF (110 mL) was added TBAF (1 M solution in THF,
108 mL, 108 mmol) and the reaction was stirred at room
temperature for 2 h. At that point MeOH (110 mL) and
K2CO3 (15.0 g, 108 mmol) were added and the reaction
was continued to stir at room temperature for an additional
12 h. The reaction was quenched by the addition of
NH4Cl, filtered through a pad of Celite, dried (Na2SO4),
and concentrated. The crude triol was passed through a short
plug of silica gel using EtOAc as eluent then concentrated.
The resulting yellow oil was diluted with DMF (130 mL)
4.1.32. Synthesis of sulfonamidoethylthioglycoside 64.
The iodosulfonamide (8.5ꢀg) was suspended in DMF
(5 mL) and cooled to ꢁ40 C. To this solution was added
a mixture of ethanethiol (2.5 mL, 33.7 mmol) and LiHMDS
(1 M solution in THF, 16.95 mL, 16.95 mmol) in DMF
(30 mL). The reaction was warmed to room temperature
then stirred for 12 h. At that point, the reaction was diluted
with saturated aq NaHCO3 (75 mL) and washed with EtOAc
(3ꢂ500 mL). The organic layer was dried over Na2SO4 and
concentrated under a stream of N2. The residue was purified
by silica gel chromatography (hexane/EtOAc¼10:1) to af-
ford 64 (6 g, 4.24 mmol, 75% over two steps) as a white
glass. [a]2D5 ꢁ33.6 (c¼0.5, CHCl3); IR (thin film): 3261,
ꢀ
then cooled to 0 C and NaH (60% suspension in oil,
2.92 g, 72.9 mmol) was added in a few portions. The reac-
ꢀ
tion was maintained at 0 C for 30 min then benzyl bromide
(12.8 mL, 108 mmol) was added drop wise and the reaction
was stirred at room temperature for 14 h. The reaction was
ꢀ
cooled to 0 C and quenched by drop wise addition of
1
MeOH. The solution was diluted with saturated aq NaHCO3
(100 mL) and washed with EtOAc (3ꢂ300 mL). The organic
layer was dried over Na2SO4 and concentrated in vacuo. The
residue was purified by silica gel chromatography (hexane/
EtOAc¼10:1) to afford the H-type trisaccharide (9.05 g,
7.71 mmol, 85%) as a white foam. [a]2D5 ꢁ22.0 (c¼2.7,
3029, 2869, 1453, 1326, 1098 cmꢁ1; H NMR (500 MHz,
CDCl3): d¼7.38–7.05 (m, 40H), 5.70 (d, J¼3.7 Hz, 1H),
4.95 (m, 2H), 4.92–4.33 (m, 21H), 3.96 (m, 3H), 3.73 (m,
1H), 3.70–3.42 (m, 9H), 3.02 (m, 2H), 2.71 (m, 2H), 1.30
(t, J¼7.4 Hz, 3H), 1.06 (d, J¼6.5 Hz, 3H), 1.06 (m, 2H),
0.02 (s, 9H); 13C NMR (125 MHz, CDCl3): d¼138.8,
138.7, 138.6, 138.4, 138.1, 138.0, 137.9, 128.6, 128.4,
128.4, 128.3, 128.2, 128.2, 128.1, 128.1, 128.0, 127.9,
127.7, 127.6, 127.5, 127.5, 127.3, 127.3, 127.2, 127.2,
126.1, 101.5, 97.4, 83.9, 83.6, 81.2, 80.2, 79.2, 77.8, 76.3,
75.7, 74.7, 74.5, 73.7, 73.4, 73.3, 73.2, 72.7, 72.4, 72.1,
70.9, 68.9, 68.1, 66.3, 57.8, 51.4, 23.9, 16.7, 14.9, 10.4,
ꢁ1.9; HRMS (FAB) calcd for C81H97NO15S2SiNa:
1438.5966, found: 1438.5990.
1
CHCl3); IR (thin film): 3029, 2869, 1453, 1098 cmꢁ1; H
NMR (500 MHz, CDCl3): d¼7.42–7.01 (m, 40H), 6.28 (d,
J¼6.3 Hz, 1H), 5.69 (d, J¼2.9 Hz, 1H), 4.94 (d,
J¼11.6 Hz, 1H), 4.87–4.80 (m, 5H), 4.60–4.38 (m, 14H),
4.23 (m, 1H), 4.14 (m, 1H), 4.04–3.95 (m, 4H), 3.80 (m,
1H), 3.72–3.57 (m, 7H), 1.19 (d, J¼6.4 Hz, 3H); 13C
NMR (125 MHz, CDCl3): d¼143.9, 139.0, 138.9, 138.5,
138.4, 138.3, 138.0, 137.9, 137.7, 128.4, 128.4, 128.3,
128.3, 128.2, 128.2, 128.1, 128.1, 128.0, 128.0, 127.9,
127.8, 127.7, 127.6, 127.6, 127.4, 127.4, 127.3, 127.2,
126.3, 101.3, 99.2, 97.0, 84.5, 79.5, 78.1, 75.5, 75.3, 74.7,
74.4, 73.6, 73.5, 73.2, 72.8, 72.6, 72.0, 71.3, 71.1, 70.3,
70.1, 68.7, 67.7, 66.2, 16.6; HRMS (FAB) calcd for
C74H78O13Na: 1197.5340, found: 1197.5389.
4.1.33. Synthesis of ethylthioglycoside amine. The sulfo-
namidoethylthioglycoside 64 (2.80 g, 1.97 mmol) was sus-
pended in DMF (15 mL) and CsF (1.5 g, 9.86 mmol) was
ꢀ
added. The reaction was stirred at 100 C for 5 d then cooled
to room temperature, diluted with saturated aq NaHCO3
(75 mL), and washed with EtOAc (3ꢂ75 mL). The organic
layer was dried over Na2SO4 and concentrated in vacuo.
The residue was purified by silica gel chromatography (hex-
ane/EtOAc¼5:1) to afford the free amine (1.63 g,
1.30 mmol, 65%) as a white glass. IR (thin film): 3382,
4.1.31. Synthesis of iodosulfonamide. To a solution of the
H-type 2 trisaccharide glycal (6.64 g, 5.65 mmol) in
CH2Cl2 (160 mL) was added 2-(trimethylsilyl)ethanesulfo-
˚
namide (1.53 g, 8.45 mmol) and powdered 4 A molecular
sieves (5 g, freshly flame-dried). The mixture was ꢀstirred
at room temperature for 20 min and then cooled to 0 C. Io-
donium-di-sym-collidine perchlorate (5.3 g, 11.32 mmol)
was added in one portion and the reaction was stirred for
1 h and subsequently diluted with CH2Cl2 (100 mL) and fil-
tered. The filtrate was washed with saturated aq Na2S2O3
(2ꢂ75 mL), saturated aq CuSO4 (1ꢂ75 mL), saturated aq
Na2S2O3 (1ꢂ75 mL), and brine (1ꢂ75 mL). The organic
layer was dried over Na2SO4 and concentrated in vacuo.
The residue (8.5 g) was used without further purification.
[a]2D5 ꢁ20.2 (c¼0.7, CHCl3); IR (thin film): 3260, 3029,
3029, 2868, 1453, 1099 cmꢁ1 1H NMR (500 MHz,
;
CDCl3): d¼7.32–7.07 (m, 40H), 5.71 (d, J¼3.7 Hz, 1H),
5.22 (d, J¼10.3 Hz, 1H), 4.95 (d, J¼11.6 Hz, 1H), 4.85 (d,
J¼11.3 Hz, 1H), 4.77–4.20 (m, 18H), 4.05–3.25 (m, 14H),
2.90 (m, 1H), 2.72 (m, 2H), 1.32 (t, J¼7.5 Hz, 3H), 1.21
(d, J¼6.5 Hz, 3H); 13C NMR (125 MHz, CDCl3):
d¼138.8, 138.6, 138.5, 138.4, 138.3, 138.0, 137.9, 128.5,
128.3, 128.3, 128.2, 128.1, 128.1, 128.0, 127.9, 127.8,
127.7, 127.6, 127.5, 127.4, 127.3, 127.2, 127.1, 127.0,
126.1, 100.7, 97.3, 86.2, 84.5, 84.1, 79.1, 77.8, 75.6, 75.5,
75.2, 74.7, 74.6, 73.5, 73.3, 73.1, 72.6, 72.2, 72.1, 70.8,
68.3, 68.0, 66.3, 55.5, 23.9, 16.7, 15.4; HRMS (FAB) calcd
for C76H85NO13SNa: 1274.5639, found: 1274.5596.
1
2949, 1453, 1331, 1139, 1068 cmꢁ1; H NMR (500 MHz,
CDCl3): d¼7.37–7.00 (m, 40H), 5.65 (d, J¼3.8 Hz, 1H),
5.09 (t, J¼10.4 Hz, 1H), 4.92–4.17 (m, 21H), 4.15 (m,
3H), 4.00 (m, 1H), 3.89 (m, 2H), 3.77–3.62 (m, 5H), 3.51
(m, 4H), 1.15 (d, J¼6.4 Hz, 3H), 1.05 (m, 2H), ꢁ0.04 (s,
4.1.34. Synthesis of phthalamidoethylthioglycoside 55. To
a solution of the ethylthioglycoside amine (800 mg,

Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
4975
0.639 mmol) in pyridine (5 mL) was added phthalic anhy-
dride (142 mg, 0.958 mmol) and the reaction was stirred at
room temperature for 45 min. Acetic anhydride (1.2 mL,
12.7 mmol) was added and the reaction was stirred at
(20 mL) and brine then dried (Na2SO4) and concentrated.
The residue was purified over silica gel (EtOAc/
hexane¼1:4, then EtOAc/toluene¼4:1) to afford the nona-
saccharide glycal 65 (440 mg, 0.111 mmol, 62%) as a white
foam. H NMR (500 MHz, CDCl3): d¼7.68 (d, J¼7.4 Hz,
1
ꢀ
85 C for 5 h. The solution was cooled to room temperature,
diluted with 50 mL of saturated aq NaHCO3, and washed
with EtOAc (3ꢂ50 mL). The organic layer was dried over
Na2SO4 and concentrated in vacuo. The residue was purified
by silica gel chromatography (hexane/EtOAc¼10:1) to af-
ford 55 (860 mg, 0.626 mmol, 98%) as a white foam. [a]D25
ꢁ37.5 (c¼0.4, CHCl3); IR (thin film): 3028, 2926, 2867,
2H), 6.70–7.50 (m, 115H), 6.60 (d, J¼8.6 Hz, 2H), 6.19
(d, J¼6.1 Hz, 1H), 5.20 (d, J¼8.0 Hz, 1H), 4.99 (d,
J¼8.0 Hz, 1H), 4.98 (s, 1H), 4.79 (s, 1H), 4.50–4.78 (m,
17H), 4.25–4.49 (m, 48H), 3.20–4.20 (m, 70H), 1.98 (s,
3H); 13C NMR (125 MHz, CDCl3): d¼170.9, 166.9, 159.1,
144.7, 141.8, 139.3, 138.8, 138.7, 138.6, 138.5, 138.2,
128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1,
128.0, 127.9, 127.8, 127.7, 127.6, 123.5, 114.0, 103.2,
100.7, 100.6, 98.2, 97.1, 80.3, 78.3, 75.3, 75.2, 74.6, 74.5,
73.7, 73.6, 73.5, 73.0, 72.7, 70.8, 67.8, 55.9, 55.3, 40.8,
40.7, 21.4; LRMS (FAB) calcd for C230H233Cl2N3O51S:
3954, found: 2000 (M+2Na)/2e.
1801, 1714, 1453, 1100 cmꢁ1
;
1H NMR (500 MHz,
CDCl3): d¼8.00 (m, 1H), 7.67 (m, 3H), 7.33–7.04 (m,
35H), 6.88–6.75 (m, 5H), 5.70 (d, J¼3.8 Hz, 1H), 5.19 (d,
J¼10.0 Hz, 1H), 4.95 (d, J¼11.6 Hz, 1H), 4.86 (d,
J¼12.0 Hz, 1H), 4.75–4.62 (m, 14H), 4.04 (m, 2H), 3.92
(m, 1H), 3.86 (dd, J¼2.5, 10.2 Hz, 1H), 3.77 (m, 2H), 3.58
(dd, J¼2.7, 9.7 Hz, 1H), 3.47 (m, 4H), 2.69 (m, 2H), 1.33
(d, J¼6.5 Hz, 3H), 1.19 (t, J¼7.4 Hz, 3H); 13C NMR
(125 MHz, CDCl3): d¼167.9, 167.5, 138.9, 138.8, 138.7,
138.6, 138.5, 138.3, 138.1, 138.0, 133.9, 133.8, 131.7,
131.6, 128.4, 128.4, 128.3, 128.2, 128.1, 128.0, 128.0,
127.9, 127.8, 127.7, 127.6, 127.5, 127.5, 127.4, 127.4,
127.3, 127.2, 127.1, 126.8, 126.1, 123.4, 123.2, 100.7,
97.5, 83.9, 81.1, 80.3, 79.2, 78.1, 77.7, 77.7, 76.4, 75.6,
74.8, 74.5, 74.4, 73.5, 73.4, 73.1, 72.5, 72.3, 72.2, 70.8,
68.3, 66.4, 54.7, 23.7, 16.7, 15.0; HRMS (FAB) calcd for
C84H87NO15SNa: 1404.5964, found: 1404.5696.
4.1.37. Synthesis of dechloroacetylated nonasaccharide
66. To a solution of the nonasaccharide glycal 65
(430 mg, 0.11 mmol) in toluene (2 mL) and ethanol
(30 mL) were added thiourea (42 mg, 0.55 mmol) and solid
NaHCO3 (140 mg, 1.66 mmol). The mixture was heated at
ꢀ
75 C for 15 h, cooled to room temperature, and concen-
trated. The residue was purified over silica gel (toluene/
EtOAc¼4:1) to afford diol 66 (410 mg, 0.11 mmol,
>99%) as a white foam. 1H NMR (500 MHz, CDCl3):
d¼7.80 (d, J¼8.4 Hz, 2H), 6.80–7.60 (m, 115H), 6.70 (d,
J¼8.5 Hz, 2H), 6.30 (d, J¼6.2 Hz, 1H), 5.35 (d,
J¼7.9 Hz, 1H), 5.25 (d, J¼8.3 Hz, 1H), 5.15 (s, 1H), 4.95
(s, 1H), 4.00–4.93 (m, 71H), 3.20–3.95 (m, 52H), 2.95 (br
d, 3H), 2.85 (br, 1H), 2.74 (d, J¼6.0 Hz, 1H), 2.20 (s,
1H), 2.10 (s, 1H), 2.08 (s, 3H); 13C NMR (125 MHz,
CDCl3): d¼170.9, 159.2, 144.8, 141.9, 139.3, 139.2,
138.9, 138.8, 138.6, 138.4, 128.9, 128.8, 128.7, 128.6,
128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8,
127.7, 127.6, 127.5, 125.7, 123.5, 114.2, 103.8, 103.4,
100.8, 100.6, 99.4, 97.2, 81.0, 80.8, 78.4, 76.3, 75.3, 75.2,
74.6, 74.5, 74.4, 73.7, 73.6, 73.5, 73.4, 73.3, 73.0, 72.8,
70.9, 69.8, 68.5, 55.9, 55.4, 21.4; LRMS (FAB) calcd for
C226H231N3O49S: 3802.5, found: 1925 [M+2Na]/2, 1936
[M+2Cl]/2.
4.1.35. Synthesis of core mannose pentasaccharide accep-
tor 51. To a solution of glycal 39 (90 mg, 41 mmol) in THF
(3 mL) was added TBAF (1 M solution in THF, 0.4 mL,
0.4 mmol) and the reaction was stirred at room temperature
for 48 h. At that point, the reaction mixture was concentrated
and purified over silica gel (hexane/EtOAc¼1:1) to afford 51
(63 mg, 31.5 mmol, 77%) as a white foam. Rf 0.42 (EtOAc/
hexane¼2:1); 1H NMR (500 MHz, CDCl3): d¼7.68 (d,
J¼7.4 Hz, 2H), 7.08–7.32 (m, 57H), 6.72 (d, J¼8.6 Hz,
2H), 6.21 (d, J¼6.1 Hz, 1H), 5.19 (d, J¼2.9 Hz, 1H), 5.03
(s, 1H), 4.87 (s, 1H), 4.20–4.72 (m, 29H), 3.30–3.95 (m,
32H), 3.22 (m, 1H), 2.98 (br d, J¼6.0 Hz, 1H), 2.19 (s,
3H), 13C NMR (125 MHz, CDCl3): d¼159.9, 145.0, 142.0,
139.0, 138.8, 138.5, 138.4, 130.4, 129.1, 129.0, 128.9,
128.8, 128.6, 128.5, 128.4, 128.3, 128.3, 128.2, 128.1,
128.1, 114.5, 102.3, 101.1, 100.9, 100.6, 98.1, 80.3, 80.2,
76.5, 75.6, 75.3, 75.2, 74.8, 73.9, 73.8, 73.6, 72.6, 72.1,
72.0, 71.0, 69.4, 68.5, 66.5, 60.9, 58.3, 55.8, 21.7; LRMS
(FAB) calcd for C116H125NO27S: 1996, found: 2019
(M+Na); 2031 (M+Cl) and 1995 (MꢁH).
4.1.38. Synthesis of pentadecasaccharide glycal 67. The
H-type 2trisaccharidedonor 55 (1.0 g, 0.72 mmol)and nona-
saccharide diol acceptor 66 (410 mg, 0.108 mmol) were
azeotropically dried with toluene (3ꢂ20 mL) and then dried
under high vacuum for 15 h. The residue was dissolved in
CH2Cl2 (10 mL), ether (20 mL), and 2,6-di-tert-butylpyri-
dine (DTBP, 658 mL, 2.94 mmol, 27 equiv) and then freshly
˚
4.1.36. Synthesis of nonasaccharide glycal 65. Lactos-
amine donor 60 (1.18 g, 1.12 mmol) and pentasaccharide
acceptor 51 (0.37 g, 0.18 mmol) were combined and
azeotropically dried with toluene (3ꢂ20 mL). The residue
was dissolved in CH2Cl2 (10 mL) and 2,6-di-tert-butyl-
dried 4 A molecular sieves (3 g) were added. After stirring
for 30 min at room temperature, the mixture was cooled to
ꢀ
0 C and treated with MeOTf (306 mL, 2.71 mmol). The re-
action was slowly warmed to room temperature and stirred
for 48 h. The reaction was quenched with saturated aq
NaHCO3 and then filtered through a pad of Celite. The fil-
trate was washed with saturated aq NaHCO3 and brine
then dried (Na2SO4) and concentrated. The residue was pu-
rified over silica gel (toluene/EtOAc¼4:1) to afford glycal
67 (560 mg, 0.09 mmol, 78%) as a white foam. Rf 0.5 (tolu-
ene/EtOAc¼4:1); 1H NMR (500 MHz, CDCl3): d¼7.70 (d,
J¼8.2 Hz, 2H), 6.60–7.60 (m, w200H), 6.48 (d, J¼8.5 Hz,
2H), 6.25 (d, J¼6.2 Hz, 1H), 5.65 (s, 2H), 5.30 (d,
˚
pyridine (1 mL, 4.46 mmol) then freshly dried 4 A MS
(1.0 g) was added. The resulting slurry was stirred at room
ꢀ
temperature for 30 min then cooled to 0 C. MeOTf
(0.41 mL, 3.63 mmol) was added drop wise, and the mixture
was slowly warmed to room temperature then stirred for 2 d.
The reaction was quenched with saturated aq NaHCO3
(5 mL), diluted with EtOAc (100 mL), and filtered through
Celite. The filtrate was washed with saturated aq NaHCO3

4976
Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
J¼6.8 Hz, 2H), 4.75–5.05 (m, 14H), 4.50–4.70 (m, 20H),
4.05–4.50 (m, 92H), 3.60–4.00 (m, 50H), 3.00–3.60 (m,
70H), 1.88 (s, 3H), 1.38 (d, J¼6.3 Hz, 6H); 13C NMR
(125 MHz, CDCl3): d¼172.5, 169.5, 168.8, 159.5, 144.9,
142.8, 139.8, 139.7, 138.6, 138.5, 138.3, 136.5, 134.5,
124.8, 116.5, 114.5, 104.5, 103.7, 102.8, 101.5, 98.0, 97.5,
96.0, 84.0, 78.0, 77.4, 76.5, 76.4, 76.3, 76.2, 75.6, 75.4,
75.2, 74.8, 74.6, 74.5, 74.2, 74.1, 73.7, 72.5, 71.9, 71.7,
68.7, 67.2, 66.8, 57.5, 54.8, 37.0, 31.2, 22.0, 18.7; LRMS
(FAB) calcd for C390H393N5O79S: 6442, found: 2171
(M+3Na)/3; 1634 (M+4Na)/4.
(d, J¼5.5 Hz, 6H); 13C NMR (500 MHz, CDCl3, selected
peaks): d¼168.9, 137.7, 137.4, 137.0, 136.8, 136.7, 127.9,
127.4, 127.3, 127.2, 127.1, 127.0, 126.9, 126.8, 126.6,
126.5, 126.4, 126.1, 124.4, 112.7, 101.6, 96.5, 82.9, 73.8,
73.5, 73.4, 72.4, 72.1, 72.0, 71.5, 71.3, 71.2, 67.4, 67.1,
65.3, 24.7, 22.0, 20.4, 15.8; LRMS calcd for
C368H395N5O76S: 6136, found: 2046 (M+3H)/3e, 2068
(M+3Na)/3e.
4.1.41. 15-mer Iodosulfonamidation product. The penta-
decasaccharide glycal 71 (130 mg, 21.2 mmol) and benzene-
sulfonamide (35 mg, 0.222 mmol) were mixed and
azeotropically dried with toluene (3ꢂ5 mL). After addition
4.1.39. Synthesis of deprotected pentadecasaccharide 50.
To a solution of 67 (100 mg, 0.02 mmol) in toluene (2 mL)
and EtOH (15 mL) was addedꢀethylenediamine (0.70 mL).
The mixture was heated at 80 C for 24 h then the volatiles
were removed at reduced pressure. The residue was purified
over silica gel (MeCl2/MeOH¼20:1) to yield the free amine
intermediate (90 mg).
˚
of freshly dried 4 A MS (500 mg) and CH2Cl2 (8 mL), the
ꢀ
suspension was cooled to ꢁ15 C. Under an atmosphere
of argon, iodonium-di-sym-collidine perchlorate (80 mg,
0.17 mmol) in CꢀH2Cl2 (2 mL) was added and the reaction
was stirred at 0 C for 30 min. After diluting with EtOAc
(150 mL), the reaction mixture was filtered through a pad
of Celite, which was thoroughly washed with EtOAc
(20 mL). The combined filtrates were successively washed
with saturated aq Na2S2O3 (3ꢂ20 mL), saturated aq
CuSO4 (2ꢂ20 mL), saturated aq Na2S2O3 (20 mL), and
brine (20 mL) then dried (Na2SO4) and concentrated in va-
cuo. The residue was purified over silica gel (4% MeOH in
CH2Cl2) to afford the intermediate iodosulfonamide
A solution of free amine (42 mg, 0.01 mmol) in THF (2 mL)
was cooled to 78 C. Gaseous NH3 (w10 mL) was con-
ꢀ
densed into the reaction vessel and solid sodium (95 mg,
4.1 mmol) was added under vigorous stirring. The resulting
ꢀ
blue solution was stirred at ꢁ78 C for 1 h then quenched
with solid NH4Cl and MeOH (5 mL) until the solution
turned clear. The resulting mixture was stirred for additional
1
(100 mg, 15.6 mmol, 74%) as a slightly yellow foam. H
ꢀ
60 min at ꢁ78 C then warmed to room temperature. The
NMR (500 MHz, CDCl3): d¼6.80–7.60 (m, w190H), 6.60
(d, J¼6.8 Hz, 2H), 5.60 (s, 2H), 5.20–5.40 (bm, 5H),
3.20–5.00 (m, w100H), 1.85 (s, 3H), 1.83 (s, 3H), 1.62 (s,
3H), 1.61 (s, 3H), 1.58 (s, 3H), 1.38 (s, 3H), 1.20 (d,
J¼6.4 Hz, 6H), LRMS (FAB) calcd for C374H401IN6O78S2:
6414, found: 2162 (M+3Na)/3e.
NH3 was removed under stream of nitrogen then further
dried under high vacuum for 2 h. Acetic anhydride (1 mL),
pyridine (1 mL), and DMAP (20 mg) were added to the
dry glycan. The mixture was stirred at room temperature
for 24 h then the volatiles were removed in vacuo. The resi-
due was purified over silca gel (MeCl2/MeOH¼20:1). To the
residue was added MeOH (0.2 mL) and NaOMe (10 mL,
0.5 M in MeOH). The reaction was stirred at room temper-
ature overnight then concentrated in vacuo. The residue
was purified by size-exclusion chromatography (LH-20 col-
umn) using MeOH/H2O (1:1) as eluent to afford pentadeca-
4.1.42. Hydrolysis of iodosulfonamide: synthesis of 2-sul-
fonamido-1-hydroxy pentadecasaccharide 72. Procedure
A: To a solution of iodosulfonamide (100 mg, 15.6 mmol)
in THF (2 mL) was added saturated aq K2CO3 (2 mL) and
the reaction was stirred at room temperature for 15 h. The re-
action mixture was diluted with EtOAc (100 mL) and then
washed with saturated aq NH4Cl (10 mL) and brine
(10 mL), dried over Na2SO4 and concentrated. Chromatog-
raphy over silica gel (hexane/EtOAc¼1:1/1:5) afforded
the desired hydrolysis product 72 (60 mg, 9.5 mmol, 60%)
as a white foam.
1
saccharide 50 as a white powder (5.5 mg 30%). H NMR
ꢀ
(500 MHz, D2O, 40 C): 6.55 (d, J¼6 Hz, 1H), 5.40 (s,
2H, 2Fuc-1), 5.25 (s, 1H), 5.05 (s, 1H), 4.50–4.95 (m,
17H), 4.45 (br s, 1H), 4.18–4.40 (m, 6H), 3.60–4.20 (m,
78H), 2.25 (s, 3H), 2.20 (m, 12H), 1.35 (m, 6H); LRMS
(FAB) calcd for C100H165N5O72: 2589, found: 1317
(M+2Na)/2, 1329 (M+2Cl)/2.
Procedure B: A solution of the iodosulfonamꢀide (60 mg,
9.35 mmol) in THF (2 mL) was cooled to ꢁ78 C and then
LiHMDS (1 M solution in THF, 60 mL, 60 mmol) and AgOTf
(4 mg, 15.6 mmol) in H2O (1 mL) were added. The resulting
mixture was allowed to warm gradually to room temperature
and stirred for 15 h. Upon complete consumption of starting
material, saturated aq NaHCO3 (5 mL) was added. The reac-
tion was diluted with EtOAc (100 mL) and washed with
brine (10 mL) then dried (Na2SO4) and concentrated. Chro-
matography over silica gel afforded 72 (38 mg, 6.0 mmol,
4.1.40. Synthesis of acetylated 15-mer glycal 71. To a solu-
tion of glycal 67 (53 mg, 8.22 mmol) in ethanol (15 mL) and
toluene (2 mL) was added ethylenediamine (0.26 mL,
ꢀ
3.88 mmol). The resulting mixture was heated at 85 C for
2 d then cooled and concentrated. The residue was stirred
at room temperature with acetic anhydride (1 mL,
10.59 mmol), pyridine (2 mL, 25 mmol), and DMAP
(5 mg, 0.04 mmol) for 15 h and then concentrated. The res-
idue was purified over silica gel (CHCl3/MeOH¼20:1, con-
taining 1% Et3N) to afford the N-acetylated product 71
(43 mg, 7.19 mmol, 85%) as a white foam. 1H NMR
(500 MHz, CDCl3): d¼8.20 (d, J¼8.0 Hz, 2H), 7.20–7.60
(m, w180H), 6.83 (d, J¼8.5 Hz, 2H), 6.54 (d, J¼8.0 Hz,
1H), 5.90 (d, J¼2.0 Hz, 2H), 5.65 (d, J¼6.0 Hz, 1H), 5.50
(m, 3H), 3.50–5.30 (m,w170H), 2.17 (s, 3H), 2.10 (s, 3H),
1.87 (s, 3H), 1.82 (s, 3H), 1.64 (s, 3H), 1.62 (s, 3H), 1.45
1
63%). H NMR (500 MHz, CDCl3): d¼7.87 (d, J¼2.0 Hz,
2H), 7.75 (d, J¼2.0 Hz, 2H), 6.80–7.30 (m, w190H), 6.61
(d, J¼6.8 Hz, 2H), 6.60 (d, J¼3.0 Hz, 2H), 3.20–5.00 (m,
w105H), 1.91 (s, 3H), 1.85 (s, 3H), 1.58 (s, 3H), 1.55 (s,
3H), 1.53 (s, 3H), 1.46 (s, 3H), 1.20 (d, J¼6.0 Hz, 6H);
LRMS calcd for C374H402N6O79S2: 6306, found: 2125
(M+3Na)/3e.

Z.-G. Wang et al. / Tetrahedron 62 (2006) 4954–4978
4977
4.1.43. Global Birch-type deprotection: synthesis of 1-hy-
droxy-pentadecasaccharide 73. A solution of 72 (62 mg,
(d, J¼9.6 Hz, 1H, NH), 8.52 (d, J¼7.2 Hz, 2H, 2NH),
8.42 (d, J¼5.6 Hz, 2H, 2NH), 8.36 (d, J¼5.6 Hz, 1H,
NH), 8.35 (d, J¼9.6 Hz, 1H, NH), 8.34 (d, J¼12.8 Hz,
1H, NH), 8.32 (d, J¼6.4 Hz, 1H, NH), 8.31 (d, J¼8.8 Hz,
1H, NH), 8.29 (d, J¼8.8 Hz, 2H, 2NH), 8.14 (d,
J¼8.8 Hz, 2H, 2NH), 7.60 (s, 1H, NH, C-terminal), 7.24
(s, 1H, NH, C-terminal); 1H NMR (D2O, 800 MHz,
ꢀ
9.83 mmol) in THF (1 mL) was cooled to ꢁ78 C. Gaseous
NH3 (w10 mL) was condensed into the reaction vessel and
solid sodium (55 mg, 2.39 mmol) was added, under vigorous
ꢀ
stirring. The resulting blue solution was stirred at ꢁ78 C
for 1 h and then quenched with solid NH4Cl and MeOH
(5 mL). The, now colorless, solution was stirred for an addi-
ꢀ
22 C): d¼5.18 (d, J¼3.2 Hz, 2H, 2H-1Fuc), 4.99 (s, 1H,
ꢀ
tional 60 min at ꢁ78 C then warmed to room temperature.
H-1 Man), 4.91 (d, J¼9.6 Hz, 1H, NH), 4.80 (s, 1H, H-1
Man), 4.64 (s, 1H, H-1 Man), 4.60 (dd, J¼6.4, 7.2 Hz,
1H), 4.57 (d, J¼8.0 Hz, 2H, 2H-1), 4.49 (d, J¼8.0 Hz,
1H, H-1b), 4.46 (d, J¼8.0 Hz, 2H, 2H-1), 4.44 (d,
J¼8.0 Hz, 2H, 2H-1), 4.34 (d, J¼7.2 Hz, 1H, H-1), 4.33
(d, J¼7.2 Hz, H-1), 4.15–4.20 (m), 4.06–4.14 (m), 3.40–
4.00 (m), 2.85 (dd, J¼6.4, 16.1 Hz, 1H), 2.64 (dd, J¼6.7,
16.1 Hz, 1H), 2.06 (m, 1H, CH(CH3)2, 1.95 (s, 3H, Ac),
1.93 (s, 3H, Ac), 1.92 (s, 6H, 2Ac), 1.91 (s, 3H, Ac), 1.90
(d, J¼7.3 Hz, 6H, 2CH3Fuc), 1.09 (d, J¼7.7 Hz, 3H,
CH3), 0.82 (d, J¼5.9 Hz, 3H, CH3), 0.81 (d, J¼6.4 Hz,
6H, 2CH3), 0.77 (d, J¼8.0 Hz, CH3); LRMS calcd for
C126H211N13O81: 3202, found: 3225 (M+Na), 1624
(M+2Na)/2e, 1637 (M+2Cl)/2 in FAB mode.
The NH3 was evaporated under a stream of nitrogen and then
placed under high vacuum for 2 h. The resulting residue was
dissolved in MeOH (1 mL) and stirred with acetic anhydride
(0.2 mL, 211 mmol) at room temperature for 2 h. Upon com-
pletion of the reaction, the mixture was purified via size-ex-
clusion chromatography. The volatiles were removed at
reduced pressure until the residue exhibited constant weight
to give the desired saccharide 73 (15 mg, 5.64 mmol, 57%).
1
ꢀ
H NMR (D2O, 500 MHz, 24 C): d¼5.32 (s, 2H, 2H-1Fuc),
5.20 (s, 1H, H-1 Man), 5.12 (s, 1H, H-1 Man), 4.96 (s, 1H,
H-1 Man), 4.70 (d, J¼8.2 Hz, 2H, 2H-1), 4.59 (d, J¼7.6 Hz,
2H, 2H-1), 4.55 (d, J¼7.8 Hz, 3H, 3H-1), 4.47 (d, J¼8.0 Hz,
2H, 2H-1), 4.11–4.24 (m, 5H), 3.49–3.99 (m, br, w85H),
2.09 (s, 9H, 3Ac), 2.05 (s, 9H, 3Ac), 1.24 (d, J¼6.4 Hz,
6H, 2CH3). LRMS calcd for C103H170N6O74: 2664, found:
2688 (M+Na), 1355 (M+2Na)/2, 2698 (M+Cl).
Acknowledgments
4.1.44. Synthesis of pentadecasaccharide glycosylamine
74. To a room temperature solution of pentadecasaccharide
73 (15 mg, 5.64 mmol) in water (4 mL) was added a large ex-
cess of solid NH4HCO3. The saturated solution was stirred at
room temperature for 3 d. It is important to keep the solution
saturated by continually adding NH4HCO3. After complete
consumption of the starting material, the reaction mixture
was lyophilized. The white solid was redissolved in water
and again lyophilized. This procedure was repeated until
the weight remained constant. Pentadecasaccharide glyco-
sylamine 74 (15 mg, 5.64 mmol, >99%) was obtained as
a white powder. H NMR (D2O, 500 MHz, 24 C): d¼5.32
(d, J¼3.2 Hz, 2H, 2H-1Fuc), 5.13 (s, 1H, H-1 Man), 4.94
(s, 1H, H-1 Man), 4.72 (d, J¼8.0 Hz, 2H, 2H-1), 4.60 (d,
J¼8.0 Hz, 2H, 2H-1), 4.56 (d, J¼8.0 Hz, 3H, 3H-1), 4.48
(d, J¼7.8 Hz, 2H, 2H-1), 4.12–4.28 (m), 3.48–4.00 (m,
w90H), 2.09, 2.05, 2.01 (s, 15H, 5Ac), 1.95 (s, 3H, Ac),
1.25 (d, J¼6.5 Hz, 6H, 2CH3); LRMS calcd for
C102H171N7O73: 2662, found: 2684 (M+Na).
This work was supported by the National Institutes of Health
(grants CA103823 and CA28824 to S.J.D.). Postdoctoral fel-
lowship support is gratefully acknowledged by Z.-G.W. (US
Army breast cancer grant no.: DAMD17-97-1-7119), J.D.W.
(NIH CA-62948), V.Y.D. (US Army breast cancer grant no.:
DAMD17-03-1-0443), U.I. (NIH CA-88478), M.V. (NIH
CA-62948), and M.E. (Deutsche Forschungsgemeinschaft).
We gratefully acknowledge Dr. George Sukenick, Ms. Sylvi
Rusli, and Ms. Anna Dudkina of the Sloan-Kettering Insti-
tute’s NMR core facility for mass spectral and NMR spectro-
scopic analyses (SKI core grant no.: CA-08748), and Ms.
Rebecca Wilson for editorial assistance in the preparation
of this manuscript.
1
ꢀ
References and notes
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To a solution of glycosylamine 74 (10 mg, 3.75 mmol) in an-
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12.8 mL DIEA in 1 mL of DMSO, 7.34 mmol), pentapeptide
4837 (4 mg, 7.2 mmol), 1-hydroxybenzotriazole (100 mL
HOBt/DMSO, prepared from 25.2 mg HOBt in 1 mL of
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a white powder. HPLC gradient: A: 0.1% TFA/H2O, B:
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800 MHz, 22 C): d¼8.74 (d, J¼8.8 Hz, 1H, NH), 8.54

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37. Both the L-amino acid-containing peptide (48) and D-amino
acid-containing peptide (49) were synthesized by San San Yi
of the Microchemistry Laboratory, Sloan-Kettering Institute
for Cancer Research, using a peptide synthesizer and were
purified by standard reverse-phase HPLC using an H2O/
TFA–CH3CN/TFA gradient. The first C-terminal amino acid
was attached to the superacid-lable Fmoc-Rink Amide
MBHA resin. The subsequent amino acids, which were intro-
duced were protected as Thr(OtBu) and Asp(Dmab ester).
Standard TFA-cleavage, Dmab- and Fmoc-removal (2% hydra-
zine in MeOH), and acetylation of the N-terminus yielded the
desired peptides.
38. Prepared from lactal according to the procedure published by
Schaubach, R.; Hemberger, J.; Kinzy, W. Liebigs Ann. Chem.
1991, 7, 607–614.
39. Danishefsky, S. J.; Behar, V.; Randolph, J. T.; Lloyd, K. O.
J. Am. Chem. Soc. 1995, 117, 5701.
´
to as the Zemplen de-O-acetylation, see: Greene, T. W.; Wuts,
P. G. M. Protective Groups in Organic Synthesis; Wiley: New
York, NY, 1999; p 154.
22. Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277.
23. Amann, A.; Ourisson, G.; Luu, G. B. Synthesis 1987, 2002.
24. Meyers, A. I.; Comins, D. L.; Roland, D. M.; Henning, R.;
Shimizu, K. J. Am. Chem. Soc. 1979, 101, 7104.