Design, synthesis, and immunochemical characterization of a chimeric glycopeptide corresponding to the Shigella flexneri Y O-polysaccharide and its peptide mimic MDWNMHAA

Article abstract of DOI:10.1016/j.carres.2009.03.029

Two glycopeptide chimeras corresponding to the Shigella flexneri Y O-polysaccharide and its peptide mimic were designed in an attempt to improve the binding affinity by increasing the entropy of binding relative to the original octapeptide mimic of the O-

Full text of DOI:10.1016/j.carres.2009.03.029

Carbohydrate Research 344 (2009) 1412–1427
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Design, synthesis, and immunochemical characterization of a chimeric
glycopeptide corresponding to the Shigella flexneri Y O-polysaccharide
and its peptide mimic MDWNMHAA
B. Rehana Hossany a, Blair D. Johnston a, Xin Wen b, Silvia Borrelli a, Yue Yuan a, Margaret A. Johnson a,
B. Mario Pinto a,
*
a Department of Chemistry, Simon Fraser University, Burnaby, B.C., Canada V5A 1S6
b Institute of Elemento-organic Chemistry and Department of Chemical Biology, Nankai University, Tianjin 300071, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two glycopeptide chimeras corresponding to the Shigella flexneri Y O-polysaccharide and its peptide
mimic were designed in an attempt to improve the binding affinity by increasing the entropy of binding
relative to the original octapeptide mimic of the O-polysaccharide. The design was based on the X-ray
crystal structures of a monoclonal antibody SYA/J6 in complex with its cognate ligands, a pentasaccharide
corresponding to the S. flexneri Y O-polysaccharide and the octapeptide mimic, MDWNMHAA. Both chi-
Received 2 February 2009
Received in revised form 12 March 2009
Accepted 23 March 2009
Available online 11 April 2009
meric molecules consist of a rhamnose trisaccharide linked through an a- or b-thioglycosidic linkage to a
MDW moiety in which the W unit has been modified. We predicted that omission of the NMHAA moiety
would obviate the bound water molecules that provided complementarity with the antibody-combining
Dedicated, with respect, to Professor Dr.
Hans Kamerling on the occasion of his 65th
birthday
site, and the conformational restriction resulting from imposition of an a-turn at the C-terminus of the
peptide. The glycopeptides were then docked into the active site of SYA/J6 using the program AUTODOCK
Keywords:
3.0, and the structures were optimized. The best models obtained in each case showed that the chimeric
Shigella flexneri Y O-polysaccharide
Carbohydrate-mimetic peptide
Glycopeptide chimera
Molecular modeling
Synthesis
molecules, with either an
antibody. We report here the synthesis of the
a
- or b-thioglycosidic linkage, might be reasonable surrogate ligands for the
-glycopeptide employing solution and solid-phase strat-
a
egies. Immunochemical characterization indicated that the
a
-glycopeptide unfortunately did not inhibit
binding of SYA/J6 to the S. flexneri Y lipopolysaccharide.
Immunochemistry
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
carbohydrate antigens, other vaccination strategies need to be ex-
plored in order to overcome some of the limitations of carbohy-
drate vaccines.
Antigens that can stimulate both humoral and cellular immune
responses, are crucial in the development of effective vaccines
against pathogenic bacteria.1 While carbohydrates that coat the
surfaces of bacteria are capable of inducing an immune response
that can recognize whole bacteria,2–8 polysaccharide vaccines fail
to provide protection in infants, the elderly, and in immunodefi-
cient persons since the immune response induced might not in-
volve the T-helper cells of the immune system.2–11 Conversion of
some of these cell-surface polysaccharides into T-cell-dependent
antigens, by conjugation to carrier proteins, has resulted in poten-
tial vaccines that could successfully target infectious bacteria in
humans.5,8,11 However, progress in this field is challenging because
the complex structures of the polysaccharide–protein conjugates
make them difficult to be synthesized and characterized.12,13 Con-
sequently, to drive T-cell-dependent immune responses against
Molecular mimics of carbohydrates that have the potential to
raise antibodies that cross-react with the natural structures have
been identified, and there is now a growing interest in carbohy-
drate–mimetic peptides as vaccines to target cell-surface polysac-
charides of infectious bacteria.14–17 In addition to their ability to
elicit carbohydrate-binding antibody responses, these peptide
mimics should focus the immune responses on particular epitopes
since greater discrimination of the peptides for the corresponding
antibody-combining sites has been observed,18 thus minimizing
autoimmune reactions with self structures where the original
carbohydrate antigens would pose a threat as immunogens.17–19
Shigella flexneri Y is a virulent bacterium that causes bacillary
dysentery by invading the colonic mucosa.20 Screening of a
phage-displayed peptide library with an anti-carbohydrate
antibody SYA/J6, directed against the O-polysaccharide (Fig. 1a)
of S. flexneri
Y yielded the peptide sequence MDWNMHAA
(Fig. 1b).18 The X-ray structures and the thermodynamics of bind-
ing of the Fab complexes of SYA/J6 with a pentasaccharide portion
* Corresponding author. Tel.: +1 778 782 4152; fax: +1 778 782 4860.
E-mail address: bpinto@sfu.ca (B.M. Pinto).
URL: http://www.sfu.ca/chemistry/faculty/pinto.htm (B. Mario Pinto).
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.03.029

B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
1413
N
OH
O
OH
HO
HO
H
OH
HO
HO
O
HO
HO
HO
N
H
O
HO
O
O
S
N
A
OH
HO
O
M
O
N
O H
HO
H
NH
O
O
HN
O
O
O
O
H2N
O
A
OH
OH
S
O
O
O
O
N
HO
HO
OH
OH
HN
H
O
H2N
O
O
S
S
HO
N
O
H
N
O
O
-
O
H
O
O
D
O
O
N
-
NH
W
O-
O
-
AcHN
O
H3N
N
H
NH
+
O
O
O
N
NH
OH
H
O
N
S
M
+
H3N
H
+
H3N
S
1
2
Figure 1. Structures of the (a) the O-polysaccharide of Shigella flexneri Y, (b) its peptide mimic (c) the chimeric a-glycopeptide 1, and (d) the chimeric b-glycopeptide 2.
[?2)-
(1?2)-
a
a
-
L
-Rha-(1?2)-
a
-
L
-Rha-(1?3)-
a
-
L
-Rha-(1?3)-b-
D
-GlcNAc-
played in the conformational ensemble of the free peptide.14,15,17
Since the -helix adopted by NMHAA in the C-terminus of
-
L
-Rha-(1?] of the O-polysaccharide (Fig. 1a) and with the
a
octapeptide mimic, MDWNMHAA (Fig. 1b) have been studied in
detail, and interesting similarities and differences between the
two complexes have been revealed.21,22
MDWNMHAA is only restricted to the bound conformation and
not to that of the free peptide, we hypothesized that MDWNMHAA
might not lead directly to a cross-reactive response against the cor-
responding polysaccharide.14,15,17 Nevertheless, we believed it
could still be used in prime–boost strategies to strengthen the im-
mune responses already induced by the polysaccharide epitopes, as
shown with a peptide mimic of the capsular polysaccharide of
Cryptococcus neoformans.23 In order to test these hypotheses, we
synthesized protein conjugates of the octapeptide24 and recently
evaluated their immunogenicities.16 Indeed, we found that,
although the kinetics of the immune response in mice were slow,
cross-reactivity was observed, and an effective prime–boost strat-
egy was developed.16
Although the octapeptide (Fig. 1b) complements the shape of
the combining site groove much better than the pentasaccharide,
and makes greater contacts with the antibody-combining site, the
binding affinity of the peptide is only twofold higher than that of
the pentasaccharide, potentially compromising the immunogenic-
ity of the peptide.21,22 The affinity can be attributed to the consid-
erable unfavorable entropy that arises from a more ordered bound
peptide and the immobilization of several water molecules.17,22
Furthermore, for a peptide to be immunogenic, it might be neces-
sary that a sufficient population of the bound conformation be dis-
Figure 2. Structures of the Fab fragment of SYA/J6 antibody with (a) bound octapeptide MDWNMHAA, (b) bound pentasaccharide, (c) bound
b-glycopeptide 2.
a-glycopeptide 1, and (d) bound

1414
B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
We now report the design of two chimeric glycopeptides (Fig. 1)
that might serve as surrogate haptens against S. flexneri Y, and the
synthesis and immunochemical evaluation of the first candidate.
2. Results and discussion
Based on the information gained from the X-ray structures of
the antibody–pentasaccharide and antibody–octapeptide com-
plexes,21,22 and by molecular modeling, we have designed two chi-
meric molecules (Fig. 1c and d) that contain certain elements of
both the oligosaccharide and the octapeptide mimic, and this
should lead to increased binding affinity by increasing the entropy
of binding relative to the parent octapeptide. The glycopeptides 1
and 2 comprise the two fragments, MDW and the rhamnose trisac-
charide (A–B–C), derived from the parent octapeptide and penta-
saccharide, respectively, and linked as a thioglycoside; the two
glycopeptides differ in the a- or b-configuration at the thioglycos-
idic linkage (Fig. 1). We chose to retain the MDW portion of the
octapeptide to maintain the favorable hydrophobic interaction of
the W moiety within the combining site. Replacement of the
NMHAA unit specifically by the rhamnose trisaccharide (A–B–C)
in the glycopeptides 1 and 2, was done firstly to ensure that ring
C penetrates the deep hole at the bottom of the binding site, thus
obviating the engagement of water molecules that provide com-
plementarity with the antibody-combining site in binding of the
peptide MDWNMHAA, and secondly, to reduce any conformational
entropy differences that result from imposition of the a-turn at the
C-terminus of the parent octapeptide upon binding. The incorpora-
tion of a thioglycosidic linkage between the trisaccharide moiety
and the tripeptide in the glycopeptides 1 and 2 was done to in-
crease their stabilities compared to those of the corresponding O-
glycosides.25
Figure 3. Superposition of the (a)
a-glycopeptide 1 (cyan) on the parent octapep-
tide (pink), (b) -glycopeptide 1 (cyan) on the parent pentasaccharide (yellow), (c)
b-glycopeptide 2 (green) on the parent octapeptide (pink), and (d) b-glycopeptide 2
a
2.1. Molecular modeling
(green) on the parent pentasaccharide (yellow).
In order to validate the choice of the glycopeptides 1 and 2
(Fig. 1), they were each docked into the Fab fragment of SYA/J6
using AUTODOCK 3.026 starting from the coordinates of the two corre-
sponding moieties in the complexes Fab-ABCDA0 and Fab-
MDWNMHAA (Fig. 2a and b).21,22 The most favored mode of bind-
ing in each case, with the lowest docked energy, showed that both
1 and 2 could fit into the binding groove without steric clashes
(Fig. 2c and d). Superposition of the two binding modes of 1 and
2 on the parent octapeptide (Fig. 3a and c) and the parent penta-
saccharide (Fig. 3b and d) indicated that both glycopeptides 1
and 2 superimposed well on the parent ligands; furthermore, the
favorable hydrophobic interaction of the W moiety with the active
site, as well as the penetration of ring C into the deep hole at the
bottom of the binding site were preserved in both molecules. The
interaction of the W moiety with the hydrophobic pocket within
the Ab-combining site was more pronounced in the b-glycopeptide
(Fig. 2c and d). Thus, the docking experiments justify the choice of
1 and 2 as reasonable surrogate ligands for the antibody SYA/J6.
We report here the synthesis and immunochemical evaluation of
charides containing
a-(1?2)- and a-(1?3)-L-rhamnopyranosidic
linkages. In the present case (Scheme 2), the reducing terminal
rhamnose unit was introduced as a thioglycoside in order to facil-
itate later conversion to a 1-thiol via the corresponding glycosyl
bromide. Thus, the known 2,4-dibenzoylated rhamnose thioglyco-
side 629 was glycosylated at the 3-position with the rhamnose tri-
chloroacetimidate donor 728 to give the disaccharide derivative 8
in >90% yield. Selective methanolysis of the 20-O-acetate using
HCl–MeOH gave the disaccharide glycosyl acceptor 9 in only mod-
erate yield due to an unusually low selectivity for reaction of the
acetate over the benzoate esters. A second glycosylation reaction
with donor 7 gave the trisaccharide 10 in 79% yield. The benzoates
were replaced by acetates through base-catalyzed methanolysis,
followed by acetylation under standard conditions to give the acet-
ylated phenylthio trisaccharide 11, in quantitative yield for the two
steps. The phenylthio group was replaced by SH in a three-step
procedure that was carried out without purification of intermedi-
ates. Thus, the phenylthio group was first replaced by a bromide
by treatment of the thioglycoside 11 with IBr30 to yield the glycosyl
bromide 12. Nucleophilic displacement of the bromide gave the
isothiouronium salt 13 which was immediately hydrolyzed to the
the first candidate, namely the
to a more facile synthesis.
a-glycopeptide 1, which lent itself
2.2. Synthesis of the
a-glycopeptide 1
thiol 4. Compound 4 was obtained as the pure
lization of the crude /b product mixture from EtOAc–hexanes.
Analysis by 1H NMR spectroscopy indicated that the
anomer 4,
in CDCl3 did not undergo significant anomerization over several
days at ambient temperature.
a anomer by crystal-
Retrosynthetic analysis indicated that the
a
-glycopeptide 1
a
could be obtained by deprotection of the glycopeptide 3, which,
in turn, could be derived from the trisaccharide 4 and the tripep-
tide bromide 5 (Scheme 1).
a
The trisaccharide 4 was prepared by methods analogous to
The tripeptide 5 was prepared from the alcohol 14 by a bromin-
ation reaction using triphenylphosphine and carbon tetrabromide
those that we had previously developed27,28 to prepare oligosac-

B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
1415
OAc
AcO
OH
O
HO
AcO
AcO
SH
HO
O
AcO
H
N
Br
O
O
O
AcO
O
HO
BnO
O
HO
OAc
O
O
O
OAc
S
O
NH
OH
S
O
AcO
O
O
N
H
AcO
OAc
HO
O
O
O
FmocHN
O
O
H
AcO
O
H
O
S
N
OAc
OAc
BnO
O
N
4
5
O
NH
O
NH
O
N
H
H3N
N
FmocHN
+
H
S
S
1
3
Scheme 1. Retrosynthetic analysis of the a-glycopeptide 1.
SC6H5
O
NH
BzO
O
SC6H5
O
OBz
SC6H5
+
O
O
CCl3
BzO
BzO
O
O
OBz
7
a
O
c
BzO
OBz
BzO
OBz
O
O
O
OAc
OH
OBz
BzO
8 R = Ac
9 R = H
10
OBz
OR
6
BzO
b
OBz
OAc
d
SC(NH2)=NH2+ Br-
AcO
SC6H5
Br
O
SH
O
O
O
O
AcO
AcO
AcO
O
OAc
O
O
OAc
OAc
OAc
g
f
e
O
O
O
O
AcO
AcO
AcO
AcO
OAc
OAc
O
O
OAc
OAc
O
O
O
O
O
O
AcO
AcO
AcO
AcO
OAc
OAc
OAc
11
OAc
OAc
OAc
OAc
OAc
13
12
4
Scheme 2. Synthesis of the precursor 4. Reagents and conditions: TMSOTf, CH2Cl2, ꢀ40 °C?rt, >90%, (b) CH3COCl, MeOH, pyridine, 45 °C?rt, 58%, (c) 7, TMSOTf, CH2Cl2,
ꢀ40 °C?rt, 79%, (d) NaOMe–MeOH, MeOH, Ac2O, Pyr, quantitative, (e) IBr, CH2Cl2, (f) thiourea,CH3CN, reflux, and (g) Na2S2O5, CH2Cl2–H2O, reflux, 56% over three steps.
O
XHN
NH
OH
a
NH
OH
b
OH
O
NH
OH
O
O
H2N
XHN
NH
O
X-Asp(OR)-OHOR
NH
O
NH2
R= OBn, 40%
R= OBn, 69%
R= Ot Bu, 79%
L-Tyrptophanol
R= OBn, X= Boc
R= OtBu, X= Fmoc
OR
OR
X-Asp(OR)-Tryptophanol
H-Asp(OR)-Tryptophanol
c
S
S
NH
Br
N
H
O
O
d
OH
NH
NH
NH
NH
O
FmocHN
FmocHN
O
O
O
5, R= OBn
19, R= OtBu
14, R= OBn, 58%
20, R= OtBu, 85%, for the 2 steps OR
OR
Scheme 3. Synthesis of the tripepides 5 and 19. Reagents: (a) DCC, HOBt, (b) TFA, or piperidine, (c) Fmoc-Met-OH, DCC, HOBt, and (d) CBr4, PPh3, CH2Cl2.

1416
B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
(Scheme 3). The alcohol 14 was assembled using standard solu-
tion-phase peptide assembly techniques from
-tryptophanol,31
BOC-Asp(OBn)-OH, and Fmoc-Met-OH, with dicyclohexylcarbodi-
imide (DCC)–1-hydroxybenzotriazole (HOBt) as the coupling agent
(Scheme 3). The aspartate side-chain carboxyl group was protected
as a benzyl ester, while the N-terminal methionine was protected
eventually determined that this was not necessary. Implementa-
tion of a citric acid wash in the processing resulted in ring-cleaving
hydrolysis of the oxazoline to give a product that was tentatively
assigned the ester structure 17 (Fig. 4). This was much more polar
than the initial peptide impurity, and was then easily separable
from the glycopeptide by flash chromatography. The other acety-
lated carbohydrate impurity 15 was unchanged by this modified
workup, and was still not removed by flash chromatography. The
mixture was then used without further purification in the depro-
tection step.
L
as the
a-N-Fmoc derivative since it was envisioned that a single,
basic deprotection step would suffice for removal of all protecting
groups from both the peptide and trisaccharide portions of the tar-
get molecule 1.
The crude bromide 5 was then coupled with the trisaccharide
thiol 4 using a two-phase reaction (Scheme 4) with the phase-
transfer protocol that was recently reported to be effective for
the synthesis of thio-linked glycopeptide derivatives.32 In the pres-
ent application, however, the phase-transfer reaction suffered from
several competing reaction pathways and provided the glycopep-
tide 3 in low yield as mixtures with side products. If the processing
did not involve acidic conditions, such as an aq citric acid wash,
Treatment of the protected glycopeptide 3 with NaOMe in
MeOH resulted in formation of a more polar product as shown
by TLC analysis of the reaction mixture. Isolation of this material
by flash chromatography and analysis by HPLC, and by NMR and
MALDI spectroscopy, made it apparent that the product was a mix-
ture of several similar compounds, none of which appeared to be
the desired glycopeptide 1. The mass spectrum indicated that the
major components of the mixture had masses of 18 mass units less
than that of the expected glycopeptide 1. It was concluded that the
strongly basic deprotection conditions had resulted predominantly
in aspartimide formation, and that partial hydrolysis must have oc-
curred during the subsequent HPLC analysis to give mixtures com-
purification by flash chromatography gave the
a-glycopeptide 3,
which was contaminated by sizeable portions of two impurities.
One impurity was entirely made up of carbohydrate, and was ten-
tatively identified as the disulfide 15 resulting from air-oxidation
of thiol 4 (Fig. 4). The other impurity was entirely peptidic in char-
acter and was assigned to be the oxazoline derivative 16 (Fig. 4),
resulting from intramolecular displacement of the primary bro-
mide by nucleophilic attack of the oxygen atom of the neighboring
amide. These impurities could be removed by HPLC, but it was
prising both
a- and b-amido linkages between the aspartate and
tryptophan units (Scheme 5). This is a well-known side reaction33
in peptide synthesis and has often been observed when peptides
containing aspartate side-chain esters are deprotected by treat-
ment with base.
S
SH
FmocHN
O
AcO
O
S
O
OAc
NH
NH
O
O
O
AcO
a
OAc
Br
NH
NH
O
NH
NH
O
FmocHN
Low Yield
O
O
O
RO
S
AcO
OAc
OAc
O
OR
AcO
4
O
5, R=OBn
19, R=OtBu
OAc
O
OAc
O
AcO
O
3, R=OBn
18, R=OtBu
AcO
OAc
OAc
Scheme 4. Synthesis of the protected glycopeptide 3. Reagents: (a) NaHCO3, Bu4NHSO4, EtOAc–H2O, ꢁ16%, after HPLC.
S
FmocHN
O
S
Me
AcO
O
FmocHN
O
S
O
S
OAc
Me
Me
AcO
O
O
AcO
NH
NH
O
OAc
OAc
O
Me
AcO
O
t-BuO
O
Me
O
t-BuO
O
AcO
O
O
OAc
O
N
O
OAc
OAc
H2N
Me
O
AcO
OAc
OAc
HN
N
H
16
1
7
15
Figure 4. Structures of the (a) disulfide 15, (b) oxazoline derivative 16, and (c) ester 17.

B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
1417
S
O
S
NH
NH
*
H2N
NH
O
N
O
S
Trisaccharide(OAc)
NaOMe/MeOH
NH
S
FmocHN
NH
O
Trisaccharide
O
O
susceptible to racemization at *
undergoes hydrolysis in two ways
OBn
H2O
S
S
Trisaccharide
S
NH
Trisaccharide
O
NH
NH
HO
NH
NH
S
H2N
H2N
NH
O
O
O
O
O
OH
4 isomers by HPLC
Scheme 5. Aspartimide formation in the deprotection of the protected glycopeptide 3.
The aspartimide problem was eventually avoided by the prepa-
ration of the glycopeptide 18 in which the benzyl ester was re-
placed by a tert-butyl ester (OtBu), giving a protecting group for
the aspartate side chain that could be selectively removed under
acidic conditions. The glycopeptide 18 was prepared by the cou-
pling of the crude tripeptide bromide 19 with the thiol 4 (Scheme
4), using the phase-transfer protocol,32 but the glycopeptide 18
was obtained in low yield, and the side products 15 and 16 were
also detected. The bromo compound 19 was obtained by the bro-
The bromide 21 was prepared from
steps (Scheme 7). L-tryptophanol was first treated with Fmoc-suc-
L
-tryptophanol34 in two
cinimide to yield Fmoc-tryptophanol. Bromination of the Fmoc-
tryptophanol was then carried out using PPh3–CBr4 to afford the
bromide 21 in 65% yield. Compound 21 was obtained even more
efficiently (95%) when Fmoc-tryptophanol was converted into its
mesylate and was subsequently treated with lithium bromide
(Scheme 7).
The Fmoc protecting group in 22 was then removed using DBU–
octanethiol35 to form the amine 23, which was coupled to the
dipeptide 24 using N-hydroxybenzotriazole (HOBt) as coupling
agent to afford the protected glycopeptide 25, with a minor pep-
tide impurity that was readily removed by HPLC to afford the pro-
tected glycopeptide 25 (Scheme 7).
The dipeptide 24, in turn, was constructed on solid support
employing Fmoc chemistry36 and an HBTU–HOBt–DIPEA coupling
strategy37–39 as shown in Scheme 8. In this method, we chose the
protecting group N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-
3-methylbutyl]aminobenzyl ester (Dmab)40 on the side chain of
Fmoc-aspartic acid because it is stable to piperidine, one of the
requirements for the solid-phase synthesis of dipeptide 24.
Fmoc-Asp(ODmab)-OH (27) and the commercially available
Fmoc-Met-OH were used in the solid-phase synthesis, and since
a free carboxylic acid group was required at the C-terminus in
the synthesis of the dipeptide 24, a 2-chlorotrityl resin41 was
chosen as the solid support. The Fmoc-Asp(ODmab)-OH (27)
was successfully prepared in two steps starting from the
commercially available Fmoc-Asp-OtBu (Scheme 8). The Dmab
protecting group was first introduced on the side chain of
Fmoc-Asp-OtBu by reacting the amino acid with Dmab-OH in
the presence of 2,6-di-tert-butyl-4-methylpyridine (DTBMP),
HOBt, and DIC, to afford the product 26 in 71% yield. Treatment
of 26 with 50% TFA in CH2Cl2 gave the desired product 27 in
nearly quantitative yield (Scheme 8).
After cleavage from the resin with dilute acid, and purification
by column chromatography, the dipeptide 28 was obtained in
80% yield (Scheme 8). The conversion of the carboxylic acid of 28
to the more active pentafluorophenol ester 24 was done to increase
the coupling efficiency between the precursors 23 and 24.
The Fmoc protecting group on the fully protected glycopeptide
25 was then removed selectively using DBU–octanethiol35 so that
the side product dibenzofulvene that formed could be scavenged
by the octanethiol, and the complex that formed could be easily
mination of tripeptide 20, which in turn, was prepared from L-tryp-
tophanol,34 Fmoc-Met-OH, and Fmoc-Asp(OtBu)-OH amino acids,
following the same strategy that was used to prepare the tripep-
tide 14 (Scheme 3).
In the deprotection of the glycopeptide 18, when the –OtBu
group was first removed using TFA–TIPSiH, the intermediate free
aspartate carboxyl group was converted to a carboxylate salt by
subsequent NaOMe treatment. It therefore became resistant to
aspartimide formation, while the other protecting groups were
smoothly removed (Scheme 6). The selective cleavage of the aspar-
tate ester also meant that the acetylated carbohydrate disulfide
impurity 15 in the glycopeptide was then considerably less polar
than the free aspartate glycopeptide, and it could be readily sepa-
rated in the early fractions during chromatographic purification of
the partially deprotected glycopeptide. Nevertheless, after the
base-catalyzed deprotection that followed, a considerable amount
of sodium acetate (which was formed by neutralization of the reac-
tion mixture with acetic acid) did co-elute with the glycopeptide 1
fraction during the final chromatographic purification.
We next examined a more efficient synthesis of the glycopep-
tide 18. We reasoned that an alternative disconnection was desir-
able, in particular, one that avoided the presence of the aspartate
and methionine moieties in the coupling reaction with 4. Accord-
ingly, the thiol 4 was coupled with the Fmoc-tryptophanol bro-
mide derivative 21; here, the two-phase reaction proceeded
smoothly to give the glycopeptide 22 as the sole product in excel-
lent yield (Scheme 7).
a
1
18
Scheme 6. Deprotection of 18 to the
a-glycopeptide 1. Reagents: (a) (i) TFA–TIPSiH,
(ii) NaOMe–MeOH, 32%.

1418
B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
H
N
CH2OH
NH2
CH2Br
NHFmoc
CH2OH
NHFmoc
FmocHN
S
a
b
c
O
O
N
H
N
H
N
H
AcO
OAc
L-Tryptophanol
Fmoc-Tryptophanol
21
O
AcO
AcO
O
HO
O
22
AcO
OAc
S
d
Fmoc
HN
H
N
H
N
O
NH
O
O
e
Dmab O
H2N
N
H
S
S
O
O
O
O
AcO
AcO
AcO
OAc
23
OAc
O
O
AcO
AcO
AcO
AcO
25
O
HO
O
O
O
AcO
AcO
OAc
OAc
Scheme 7. Synthesis of the protected glycopeptide 25. Reagents: (a) Fmoc-OSu, NaHCO3, acetone–H2O, 95%, (b) (i) MsCl–py, (ii) LiBr, refluxed, 95% for the two steps, (c) 4,
NaHCO3, Bu4NHSO4, EtOAc–H2O, 92%, (d) DBU, octanethiol, THF, quantitative, and (e) 24, HOBt, THF, 72%.
OtBu
OH
O
O
NHFmoc
OtBu
NHFmoc
O
O
O
O
O
O
O
NHFmoc
O
O
a
b
c
NHFmoc
O
Cl
OH
O
O
(2-chlorotrityl resin)
O
O
N
H
Fmoc-Asp-OtBu
NH
NH
F
O
d
O
26
27
O
F
F
O
OH
O
NH
O
O
e
NH
O
O
O
S
F
S
F
O
O
O
HN
Fmoc
O
O
N
H
HN
Fmoc
N
24
28
H
Scheme 8. Synthesis of the dipeptide 24. Reagents: (a) Dmab-OH, DTBMP, HOBt, DIC, 71%, (b) 50% TFA in CH2Cl2, 97%, (c) DIPEA, DMF, (d) (i) 25% piperidine–DMF, (ii) Fmoc-
Met-OH, HBTU–HOBt–DIPEA, DMF, (iii) 5% TFA in CH2Cl2, 80%, and (e) pentafluorophenol, DIC, 73%.
removed in ether to give 29 (Scheme 9). Treatment of the amine 29
with NaOMe–MeOH, however, did not afford the desired glycopep-
tide 1 (Scheme 9), but resulted in the formation of the same aspar-
timide side product that was formed in the deprotection of the
glycopeptide 3 (Scheme 5).
However, when the Dmab and the acetate protecting groups in
29 were sequentially removed, using 2% hydrazine–THF to first
afford 30, followed by NaOMe–MeOH, the desired glycopeptide 1
was obtained in 68% yield, after treating the mixture first with
Rexyn 101 H+ to make the mixture slightly basic, followed by

B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
1419
OAc
AcO
OAc
O
AcO
AcO
AcO
AcO
OH
O
HO
O
O
HO
AcO
AcO
O
AcO
O
HO
AcO
O
O
OAc
S
OAc
S
HO
O
O
O
AcO
OH
S
O
O
O
HO
a
b
O
H
N
H
N
O
O
O
O
Dmab O
Dmab O
H3N
N
N
H
O
O
+
NH
NH
O
O
N
H
O
N
H
N
H
S
H2N
FmocHN
S
S
25
29
Aspartimide
Scheme 9. Aspartimide formation in the deprotection of the glycopeptide 25. Reagents: (a) DBU–octanethiol, THF, quantitative and (b) NaOMe, MeOH, 88%.
3. Experimental
OAc
O
AcO
AcO
AcO
3.1. Synthesis
O
AcO
3.1.1. General methods
O
The Fmoc amino acids that were used were purchased from
Novabiochem, and the other reagents were purchased from Aldrich
Chemical Co. DMF was freed of amines by concentrating it under
high vacuum, and it was then distilled and stored over 4 Å molec-
ular sieves, whereas the other solvents were distilled according to
standard procedures.42 The dipeptide 3 was synthesized on 2-chlo-
rotrityl resin41 (Novabiochem) (1.64 mmol/g substitution level),
using standard 9-fluorenylmethoxycarbonyl (Fmoc) chemistry36
employing the 2-[1H-Benzotriazole-1-yl]-1.1.13.3-tetramethyluro-
nium hexafluorophosphate–1-hydroxybenzotriazole (HBTU–HOBt)
coupling strategy.37–39 The Kaiser ninhydrin (5% ninhydrin in EtOH,
80% phenol in EtOH, and 2% 0.001 M aq KCN in pyridine) assay for
the amino group43 was used to monitor both the Fmoc-amino acid
coupling and the Fmoc deprotection reactions. 1D- and 2D-NMR
spectra were recorded on 400-, 500 and 600-MHz spectrometers.
Chemical shifts were referred to internal CHCl3 or external DSS
[3-(trimethylsilyl)-1-propanesulfonic acid] and were reported in
d units relative to tetramethylsilane. Coupling constants were ob-
tained from a first-order analysis of one-dimensional spectra, and
spectral assignments were based on COSY, HMQC, and TOCSY
experiments. MALDI-TOF mass spectra were obtained for samples
dispersed in a 2,5-dihydroxybenzoic acid matrix on a Perspective
Biosystems Voyager-DE instrument. High resolution mass spectra
were obtained using LSIMS (FAB), run on a Kratos Concept H dou-
ble focusing mass spectrometer at 10,000 RP and by the electro-
spray ionization method, using an Agilent 6210 TOF LC/MS high
resolution magnetic sector mass spectrometer.
OAc
S
a
b
O
29
1
AcO
O
H
N
O
O
-
O
NH
O
N
H
H3N
+
S
30
Scheme 10. Deprotection of 29 to the
THF, quantitative and (b) NaOMe–MeOH, 68%.
a
-glycopeptide 1. Reagents: (a) 2% hydrazine,
further neutralization with acetic acid, and purification by HPLC
(Scheme 10).
2.3. Immunochemistry
The binding of the glycopeptide to the monoclonal antibody
SYA/J6 was investigated by competitive inhibition ELISA studies,
using S. flexneri Y LPS as coating antigen. The purified glycopeptide
was analyzed as an inhibitor of the binding of monoclonal antibody
SYA/J6, while the LPS, the purified O-polysaccharide, and the mi-
metic-octapeptide, were used as control inhibitors. No inhibitory
activity was observed with the glycopeptide even at the highest
3.1.2. Phenyl 2-O-acetyl-3,4-di-O-benzoyl-a-L-
concentration tested (200
ride showed IC50 values <0.7
showed an IC50 value of 6.25
l
g/mL). Both the LPS and O-polysaccha-
g/mL and the mimetic-octapeptide
g/mL (data not shown). The relative
rhamnopyranosyl-(1?3)-2,4-di-O-benzoyl-1-thio-a-L-
rhamnopyranoside (8)
l
l
A mixture of the thioglycoside 629 (1.33 g, 2.86 mmol) and the
trichloroacetimidate 728 (1.52 g, 2.72 mmol) in anhyd CH2Cl2
(30 mL) was stirred with freshly activated, crushed 4 Å molecular
sieves (1.3 g) under N2 for 10 min at room temperature. The mix-
ture was cooled in a ꢀ40 °C bath, and trimethylsilyl triflate
inhibitory activities of multivalent carbohydrate ligand and mono-
valent peptide ligand differ from those obtained by microcalorime-
try for monovalent pentasaccharide and octapeptide ligands
(twofold difference);21,22 we attribute these differences to the ef-
fect of multivalent presentation of carbohydrate ligands. The rea-
son for the disappointing lack of inhibition by the glycopeptide is
not at all obvious and will require further investigation through
synthesis and immunochemical evaluation of its stereoisomer,
the b-linked glycopeptide.
(40 lL, 0.22 mmol) was added. After 15 min, the cooling bath
was removed, and the mixture was allowed to warm to room tem-
perature over a period of 40 min. Et3N (0.2 mL) was added, and the
molecular sieves were removed by filtration through Celite, with
the aid of additional CH2Cl2 (100 mL). The filtrate was washed with

1420
B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
satd NaHCO3 (30 mL), dried over MgSO4, and concentrated to a syr-
up. Flash chromatography (3:1 to 2:1 hexanes–EtOAc) gave the
disaccharide 8 (2.25 g, >90%) as a colorless, hard foam containing
approximately 10 mol % of the byproduct trichloroacetamide (1H
NMR, broad singlet at d 6.6). The mixture was used directly in
the next reaction. 1H NMR (400 MHz, CDCl3): d 8.25–7.25 (25 H,
m, Ar), 5.74 (1H, dd, J1,2 = 1.9, J2,3 = 3.3 Hz, H-2), 5.71 (1H, d, H-1),
H-100), 4.58 (1H, dq, H-5), 4.50 (1H, dd, H-3), 4.11 (1H, dq, H-50),
4.04 (1H, dq, H-500), 3.98 (1H, dd, H-20), 2.00 (3H, s, OAc), 1.34
(3H, d, J5,6 = 6.3 Hz, H-6), 1.23 (3H, d, J5 ,6 = 6.3 Hz, H-60), 1.11,
0
0
(3H, d, J5 ,6 = 6.3 Hz, H-600). 13C NMR (100 MHz, CD2Cl2): d 169.6
00 00
(1C, OAc), 166.3–165.3 (6C, 6 OBz), 133.9–128.4 (42C, Ar), 100.9
(1C, C-10), 99.5 (1C, C-100), 86.2 (1C, C-1), 76.8 (1C, C-20), 76.3 (1C,
C-3), 74.1 (1C, C-2), 73.8 (1C, C-4), 71.7 (1C, C-40), 71.6 (1C, C-400),
70.9 (1C, C-30), 69.9 (1C, C-300), 69.8 (1C, C-200), 68.5 (1C, C-5),
68.1 (1C, C-50), 67.8 (1C, C-500), 20.8 (1C, CH3, OAc), 17.8, 17.6 and
17.5 (3C, 3 ꢂ CH3, C-6, C-60, C-600). MALDI-TOFMS: m/z 1237.4
(M+Na). Anal. Calcd for C68H62O19S: C, 67.20; H, 5.15. Found: C,
67.11; H, 5.13.
0
0
5.63 (1H, t, J3,4 = J4,5 = 9.7 Hz, H-4), 5.47 (1H, dd, J2 ,3 = 3.4,
J3 ,4 = 9.9 Hz, H-30), 5.36 (1H, t, J4 ,5 = 9.8 Hz, H-40), 5.11 (1H, dd,
0
0
0
0
J1 ,2 = 2.0 Hz, H-20), 5.08 (1H, d, H-10), 4.54 (1H, dq, H-5), 4.42
0
0
(1H, dd, H-3), 4.06 (1H, dq, H-5), 1.95 (3H, s, OAc), 1.37 (3H, d,
J5,6 = 6.3 Hz, H-6), 1.33 (3H, d, J5 ,6 = 6.3 Hz, H-60). 13C NMR
(150 MHz, CDCl3): d 169.4 (1C, OAc), 166.0–164.7 (4C, 4 ꢂ OBz),
133.6–127.9 (30C, Ar), 99.4 (1C, C-10), 85.6 (1 C, C-1), 76.6 (1C, C-
3), 73.8 (1C, C-2), 73.2 (1C, C-4), 71.5 (1C, C-40), 69.9 (1C, C-20),
69.3 (1C, C-30), 68.3 (1C, C-5), 67.6 (1C, C-50), 20.6 (1C, OAc), 17.6
(1C, C-6), 17.3 (1C, C-60). MALDI-TOFMS: m/z 883.3 (M+Na).
0
0
3.1.5. Phenyl 2,3,4-tri-O-acetyl-
3,4-di-O-acetyl- -rhamnopyranosyl-(1?3)-2,4-di-O-acetyl-1-
thio- -rhamnopyranoside (11)
a-L-rhamnopyranosyl-(1?2)-
a-L
a-L
A solution of NaOMe–MeOH (1 M, 10 mL) was added to a sus-
pension of trisaccharide 10 (3.71 g, 3.05 mmol) in anhyd MeOH
(100 mL), and the mixture was stirred at room temperature until
the starting material had dissolved (ꢁ1 h). The solution was kept
in a closed flask for 20 h and was then neutralized by stirring with
Rexyn 101 H+ ion-exchange resin. The resin was removed by filtra-
tion, and the filtrate was concentrated by rotary evaporation to
give a syrupy residue. The residue was extracted by triturating
with hexanes (3 ꢂ 50 mL) to remove MeOBz. The insoluble trisac-
charide was acetylated with Ac2O (20 mL) and pyridine (40 mL)
for 3 h at rt. The excess reagents were removed by rotary evapora-
tion under high vacuum. The gummy residue was dissolved in Et2O
(150 mL) and was washed with satd NaHCO3 (50 mL), dried over
MgSO4, and concentrated to give the trisaccharide 11 as a hard
3.1.3. Phenyl 3,4-di-O-benzoyl-a-L-rhamnopyranosyl-(1?3)-
2,4-di-O-benzoyl-1-thio- -rhamnopyranoside (9)
a-L
The disaccharide 8 (8.15 g, 9.95 mmoL) was dissolved in MeOH
(250 mL), and the solution was stirred rapidly. AcCl (4 mL) was
added dropwise, and the mixture was stirred for 4 h at rt and then
at 45 °C for 3.5 h. The solution was cooled to rt and neutralized by
the dropwise addition of pyridine. The solvents were evaporated,
and the residue was partitioned between dichloromethane
(200 mL) and satd NaHCO3 solution (100 mL). The organic phase
was dried over MgSO4 and was concentrated to give a syrup. Puri-
fication by column chromatography (hexanes:EtOAc; 2:1) gave the
disaccharide hemiacetal 9 (4.54 g, 58%) as a colorless foam contain-
ing a trace of the starting 20-O-acetate, as indicated by TLC. 1H NMR
(500 MHz, CDCl3): d 8.25–7.25 (25H, m, Ar), 5.75 (1H, dd, J1,2 = 1.5,
J2,3 = 2.4 Hz, H-2), 5.71 (1H, d, H-1), 5.62 (1H, t, J3,4 = J4,5 = 9.7 Hz, H-
foam (2.72 g, quantitative). ½a D25
ꢃ
ꢀ 71 (c 1.2, CHCl3). 1H NMR
(600 MHz, CD2Cl2): d 7.51–7.29 (5H, m, Ar), 5.44–5.39 (2H, m, H-
1,H-2), 5.28 (1H, dd, J2 ,3 = 3.5, J3 ,4 = 10.0 Hz, H-300), 5.25 (1H,
dd, H-200), 5.10 (1H, t, J3,4 = J4,5 = 9.8 Hz, H-4), 5.06–4.99 (3H, m,
00 00
00 00
4), 5.42 (1H, t, J3 ,4 = J4 ,5 = 9.5 Hz, H-40), 5.37 (1H, dd, J2 ,3 = 2.9 Hz,
0
0
0
0
0
0
H-30), 5.09 (1H, d, J1 ,2 = 1.5 Hz, H-10), 4.56 (1H, dq, H-5), 4.45 (1H,
H-30, H-40, H-400), 4.96 (1H, d, J1 ,2 = 1.8 Hz, H-10), 4.79 (1H, d, H-
100), 4.27 (1H, dq, H-5), 4.10 (1H, dd, J2,3 = 3.1 Hz, H-3), 3.97 (1H,
0
0
0
0
dd, H-3), 4.08 (1H, dq, H-50), 3.98 (1H, br s, H-20), 2.05 (1H, br s, 20-
OH) 1.35 (3H, d, J5,6 = 6.4 Hz, H-6), 1.17 (3H, d, J5 ,6 = 6.4 Hz, H-60).
13C NMR (100 MHz, CDCl3): d 166.0, 165.5 (2C) and 164.9 (4C,
4 ꢂ OBz), 133.5–127.9 (30C, Ar), 101.5 (1C, C-10), 85.6 (1C, C-1),
76.1 (1C, C-3), 73.9 (1C, C-2), 73.4 (1C, C-4), 72.1 (1C, C-40), 71.2
(1C, C-20), 69.4 (1C, C-30), 68.1 (1C, C-5), 67.5 (1C, C-50), 17.6 (1C,
C-6), 17.3 (1C, C-60). MALDI-TOFMS: m/z 841.3 (M+Na). Anal. Calcd
for C46H42O12S: C, 67.46; H, 5.17. Found: C, 67.67; H, 5.11.
dq, J4 ,5 = 9.9 Hz,H-500), 3.92 (1H, dd, J2 3 = 1.9 Hz, H-20), 3.86 (1H,
0
0
00 00
0
0
dq, J4 ,5 = 9.9 Hz, H-50), 2.16, 2.13, 2.11, 2,05, 2.04, 2.03, 1.97 (each
0
0
3H, 7 s, 7 ꢂ OAc), 1.22 (3H, d, J5 ,6 = 6.3 Hz, H-600), 1.20 (3H, d,
00 00
J5,6 = 6.2 Hz, H-6), 1.19 (3H, d, J5 ,6 = 6.3 Hz, H-60); 13C NMR
(100 MHz, CD2Cl2): d 170.5–170.1 (7C, 7 ꢂ OAc), 133.7–128.3 (6C,
Ar), 100.5 (1C, C-10), 99.9 (1C, C-100), 86.2 (1C, C-1), 77.7 (1C, C-
20), 75.4 (1C, C-3), 73.1 (2C, C-2 and C-4), 71.1, 71.0 and 70.5 (3C,
C-30, C-40 and C-400), 70.1 (1C, C-20), 69.0 (1C, C-300), 68.3 (1C, C-
5), 67.8 (1C, C-50), 67.6 (1C, C-500), 21.1–20.9 (7C, 7 ꢂ OAc), 17.66,
17.59 and 17.48 (3C, 3 ꢂ CH3, C-6, C-60, C-600). MALDI-TOFMS: m/z
865.2 (M+Na). Anal. Calcd for C38H50O19S: C, 54.14; H, 5.98. Found:
C, 54.02; H, 6.24.
0
0
3.1.4. Phenyl 2-O-acetyl-3,4-di-O-benzoyl-
pyranosyl-(1?2)-3,4-di-O-benzoyl- -rhamnopyranosyl-
(1?3)-2,4-di-O-benzoyl-1-thio- -rhamnopyranoside (10)
a-L-rhamno-
a
-L
a-L
A mixture of the disaccharide 9 (4.54 g, 5.54 mmol) and the tri-
chloroacetimidate 7 (3.34 g, 5.98 mmol) in anhyd CH2Cl2 (90 mL)
was stirred with freshly activated, crushed 4 Å molecular sieves
(4 g) under N2 for 10 min at room temperature. The mixture was
3.1.6. 2,3,4-Tri-O-acetyl-
acetyl- -rhamnopyranosyl-(1?3)-2,4-di-O-acetyl-1-thio-
rhamnopyranose (4)
a-L-rhamnopyranosyl-(1?2)-3,4-di-O-
a
-L
a-L-
cooled in a ꢀ40 °C bath, and Et3N (80
lL, 0.45 mmol) was added.
After 30 min, the cooling bath was removed and the mixture was
allowed to warm to room temperature over a period of 0.5 h.
Et3N (0.3 mL) was added and the sieves were removed by filtration
through Celite, with the aid of additional CH2Cl2 (100 mL). The fil-
trate was washed with satd NaHCO3 (50 mL), dried over MgSO4,
and concentrated to a syrup. Purification by column chromatogra-
phy (20:1 toluene–EtOAc;) gave the trisaccharide 10 (5.36 g, 79%)
A solution of the trisaccharide phenyl thioglycoside 11 (2.99 g,
3.17 mmol) in CH2Cl2 (60 mL) was cooled in an ice-bath while a
1 M solution of IBr in CH2Cl2 (40 mL) was added dropwise. The
mixture was stirred for 20 min, then diluted with additional cold
CH2Cl2 (60 mL), and washed with cold 5% aq Na2S2O3 solution
(60 mL) and cold satd NaHCO3 solution (50 mL). The organic
phase was dried over MgSO4, and was then filtered and evapo-
rated to give the trisaccharide glycosyl bromide 12 as a colorless
foam. The bromide 12 was immediately dissolved in anhyd
CH3CN (40 mL) and was slowly warmed to reflux along with thio-
urea (0.41 g, 5.4 mmol). After 1.5 h at reflux temperature under a
N2 atmosphere, the mixture was cooled and concentrated by ro-
tary evaporation. This yielded the crude isothiouronium salt 13
as a mixture, together with the byproduct (diphenyl disulfide)
as a colorless, hard foam. ½a D25
ꢃ
þ 51 (c 1.1, CHCl3). 1H NMR
(600 MHz, CD2Cl2): d 8.25–7.29 (40H, m, Ar), 5.79 (1H, dd,
J1,2 = 1.8, J2,3 = 3.4 Hz, H-2), 5.73 (1H, d, H-1), 5.67 (1H, dd,
J2 ,3 = 3.5, J3 ,4 = 10.0 Hz, H-300), 5.59 (1H, t, J3,4 = J4,5 = 9.6 Hz, H-
00 00
00 00
4), 5.56 (1H, dd, J2 ,3 = 3.3, J3 ,4 = 10.1 Hz, H-30), 5.46 (1H, t,
0
0
0
0
J4 ,5 = 9.8 Hz, H-40), 5.45 (1H, dd, J1 ,2 = 1.7, Hz, H-200), 5.37 (1H, t,
0
0
00 00
J4 ,5 = 9.9 Hz, H-400), 5.23 (1H, d, J1 ,2 = 1.8 Hz, H-10), 4.60 (1H, d,
00 00
0
0

B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
1421
and excess thiourea. Without further purification, the crude prod-
uct was dissolved in a two-phase mixture of CH2Cl2 (40 mL) and
water (10 mL) containing Na2S2O5 (1.5 g). Hydrolysis of the iso-
thiouronium salt 13 over 0.5 h was brought about by rapid stir-
ring of the heterogeneous reaction mixture while refluxing
under N2. The mixture was cooled to rt and was diluted with
additional CH2Cl2 (100 mL). The organic phase was washed with
water (50 mL) and satd NaCl solution (50 mL), dried over MgSO4,
and concentrated to give a syrupy residue. Purification by chro-
TFA was removed by rotary evaporation, and the residue was par-
titioned between EtOAc (100 mL) and satd aq NaHCO3 solution.
The EtOAc phase was again washed with aq NaHCO3 solution
(30 mL) and then with satd aq NaCl solution (20 mL). The
solution was dried over MgSO4 and was evaporated to give a
colorless syrup.
The crude product H-Asp(OBn)-tryptophanol (0.730 g) was dis-
solved in dry THF (20 mL). Fmoc-Met (0.693 g, 1.87 mmol) was
added, followed by HOBt (0.253 g, 1.87 mmol) and DCC (0.386 g,
1.87 mmol). The mixture was stirred at rt for 1 h and was then fil-
tered to remove DCU. The THF was removed to leave a residue,
which was only sparingly soluble in EtOAc. Most of the material
was brought into solution by rapid stirring with a mixture of EtOAc
(150 mL) and satd aq NaHCO3 solution for 0.5 h. The EtOAc layer
was then separated and washed with 10% citric acid (30 mL), satd
NaHCO3 solution (30 mL), and satd NaCl solution (20 mL). The
solution was dried (MgSO4) and concentrated to give the crude tri-
peptide as a pale-yellow solid. Attempted purification by flash
chromatography (1:2 hexanes:EtOAc to EtOAc) resulted in only a
marginal improvement in purity with the major contaminant
(DCU) co-eluting with the product. A suitable solvent for recrystal-
lization could not be found, but eventually a jelly-like solid, with
acceptable purity, was obtained by slow cooling of a hot EtOH solu-
tion. Pumping of the gel on high vacuum gave the tripeptide 14 as a
white solid (0.810 g, 58%). 1H NMR (400 MHz, DMSO-d6): d 10.8
matography on silica gel (1:1 hexanes–EtOAc) gave an
ture of the trisaccharide glycosyl thiol 4 as a colorless syrup
(1.52 g, 56% for three steps). Selective crystallization of the ano-
a/b mix-
a
mer from EtOAc–hexanes gave pure 4 (857 mg) as colorless fine
needles. mp 202–204° C;
½
a 2D5
ꢃ
ꢀ 63 (c 1,CHCl3). 1H NMR
(600 MHz, CDCl3): d 5.50 (1H, dd, J1,2 = 1.6, J1,SH = 7.0 Hz, H-1),
5.32 (1H, dd, J2 ,3 = 3.5, J3 ,4 10.1 Hz, H-300), 5.27–5.25 (2H, m,
H-2, H-200), 5.09 (1H, t, J3,4 = J4,5 = 9.8 Hz, H-4), 5.06 (1H, t,
00 00
00
0
J3 ,4 = J4 ,5 = 9.8 Hz, H-400), 5.06–5.01 (2H, m, H-3, H-4), 4.91
00 00
00 00
(1H, d, J1 ,2 = 1.8 Hz, H-10), 4.76 (1H, d, J1 ,2 = 1.8 Hz, H-100),
0
0
00 00
4.25–4.08 (2H, m, H-3, H-5), 3.96 (1H, dq, H-500), 3.92 (1H, dd,
J2 3 = 2.1 Hz, H-20), 3.83 (1H, dq, J4 ,5 = 9.9 Hz, H-50), 2.21 (1H, d,
0
0
0
0
SH), 2.20, 2.13, 2.12, 2.07, 2.06, 2.04 and 2.00 (each 3H, 7 s,
7 ꢂ OAc), 1.22 (3H, d, J5 ,6 = 6.3 Hz, H-600), 1.20 (3H, d,
00 00
J5,6 = 6.3 Hz, H-6), 1.19 (3H, d, J5 ,6 = 6.3 Hz, H-60). 13C NMR
(150 MHz, CDCl3): d 170.3, 170.3, 170.1, 169.8, 169.8, 169.6,
169.6 (7C, 7 ꢂ OAc), 99.9 (1C, C-10), 99.6 (1C, C-100), 77.5 (1C, C-
20), 76.6 (1C, C-1), 73.8 (1C, C-200), 73.7 (1C, C3), 72.8 (1C, C-4),
70.9 (1C, C-400), 70.8 (1C, C-40) 70.02 (1C, C-30), 70.0 (1C, C-30),
68.8 (1C, C-20), 68.5 (1C, C-300), 67.9 (1C, C-5), 67.4 (1C, C-50),
67.2 (1C, C-500), 20.9, 20.8 (2C), 20.8 (3C), 20.7 (7C, 7 ꢂ OAc),
17.4, 17.4 and 17.3 (3C, 3 ꢂ CH3, C-6, C-60, C-600). MALDI-TOFMS:
m/z 789.2 (M+Na). Anal. Calcd for C32H46O19S: C, 50.12; H, 6.05.
Found: C, 50.24; H, 6.17.
0
0
(1H, br s, NH-Trp ring), 8.35 (1H, d, J ,NH = 8.0 Hz, NH- Asp),
a
7.90–7.24 (17H, m, 13 Ar-H [Fmoc and Bn], H4-, H7-Trp,
NH- Met, NH- Trp), 7.10 (1H, d, J1,2 = 1.9 Hz, H2-Trp), 7.04 (1H, br
t, J 5,6 ꢄ J6,7 ꢄ 7.5 Hz, H6-Trp), 6.95 (1H, br t, J 5,6 ꢄ J4,5 ꢄ 7.5 Hz,
H5-Trp), 5.06 (2H, br s, -OCH2- Bn), 4.68 (1H, t, J = 5.5 Hz,
-OH- Trp), 4.64 (1H, m,
and C-9H-Fmoc), 4.08 (1H, m,
3.32 (2H, m, b-CH2- Trp), 2.86 (2H, br dd, b-CH2- Trp, and b-CH2-
Asp), 2.74 (1H, dd, JA,B = 14.4, J ,B = 5.9 Hz, b-CH2- Trp), 2.66 (1H,
a-H- Asp), 4.34–4.16 (3H, m, CH2-Fmoc
a
-H- Met), 3.92 (1H, m, -H- Trp),
a
a
3.1.7. Fmoc-Met-Asp(OBn)-tryptophanol (14)
dd, JA,B = 11.1, J ,B = 8.3 Hz, b-CH2- Asp), 2.49 (2H, br t, J = 7.4 Hz,
a
L
-Tryptophanol34 (1.97 g, 10.4 mmol), BOC-Asp(OBn)-OH
-SCH2- Met), 2.01 (3H, s, -SCH3- Met), 1.84 (2H, m, b–CH2- Met).
13C NMR (100 MHz, DMSO-d6): d 171.5, 170.1 169.6 (3C, 3 ꢂ C@O),
156.1 (1C, C@O- Fmoc), 143.9–111.0 (26C, Ar), 65.7 (2C, -OCH2-
(3.23 g, 10.0 mmol), and HOBt (1.35 g, 10.0 mmol) were combined
in dry THF (60 mL). DCC (2.06 g, 10.0 mmol) was added, and the
mixture was stirred at rt for 1 h. After filtration to remove dicyclo-
hexylurea (DCU), the solvent was removed by rotary evaporation,
and the residue was partitioned between EtOAc (150 mL) and satd
aq NaHCO3 (50 mL). The organic phase was washed with 10% citric
acid (50 mL), and then with satd aq NaHCO3 (2 ꢂ 50 mL) and satd
aq NaCl (30 mL). The solution was dried over MgSO4 and was evap-
orated to an amorphous solid. The crude product was purified by
flash chromatography on silica gel (1:2 hexanes–EtOAc) to give
compound BOC-Asp(OBn)-tryptophanol as a colorless, hard foam
(3.42 g, 69%). 1H NMR (400 MHz, CDCl3): d 8.07 (1H, br s, NH-Trp
ring), 7.65 (1H, br d, J4,5 = 7.9 Hz, H4-Trp), 7.40–7.28 (6H, m, H7-
Trp and Bn Ar), 7.20 (1H, ddd, J6,7 = 8.0, J5,6 = 7. 1, J4,6 = 1.2 Hz,
H6-Trp), 7.13 (1H, ddd, J4,5 = 8.0, J5,7 = 1.1 Hz, H5-Trp), 7.06 (1H,
Fmoc and -OCH2- Bn), 61.8 (1C, -OCH2- Trp), 54.0 (1C, C
a-Met),
51.9 (1C, C -Trp), 49.6 (1C, C -Asp), 46.6 (1C, C9-Fmoc), 36.1
a
a
(1C, Cb-Asp), 31.4 (1C, t, Cb-Met), 29.5 (1C, SCH2-Met), 26.3 (1C,
Cb-Trp), 14.6 (1C, SCH3-Met). HRMS Calcd for C39H46N4O7S: m/z
749.3008. Found: m/z 749.3014.
3.1.8. Fmoc-Met-Asp(OBn)-tryptophanyl bromide (5)
The tripeptide alcohol 14 (0.157 g, 0.210 mmol) was dispersed
with stirring in CH2Cl2 (5 mL). Triphenylphosphine (0.121 g,
0.461 mmol) and CBr4 (0.163 g, 0.491 mmol) were added, which
resulted in the peptide dissolving within 10 min to give a homoge-
neous yellow solution. After a further 10 min, the solvent was
removed to yield the crude product as a sticky yellow gum. This
was purified by flash chromatography on silica gel (1:1 to 1:2 hex-
anes–EtOAc) to provide the bromide 5 as a pale-yellow solid. This
material was only of limited stability at rt and was generally used
immediately in the next step. 1H NMR (400 MHz, CDCl3): d 8.13
(1H, br s, NH-Trp ring), 7.78–7.22 (17H, m, Ar-[Fmoc and Bn],
H4-, H7-Trp, NH-Met, NH-Asp) 7.19 (1H, ddd, J6,7 = 8.0, J5,6 = 7.1,
J4,6 = 1.2 Hz, H6-Trp), 7.13 (1H, ddd, J4,5 = 8.0, J4,6 = 1.0 Hz, H5-
d, J1,2 = 2.3 Hz, H2-Trp), 6.65 (1H, br d, JNH, = 7.7 Hz, NH-Trp),
a
5.54 (1H, br d, JNH, = 7.8 Hz, NH-Asp), 5.12 and 5.07 (1H each,
a
2d, JA,B = 12.3 Hz, -OCH2- Bn), 4.47 (1H, br m,
m, -H-Trp), 3.66 (1H, dd, JA,B = 11.2, JA, = 3.8 Hz, b-CH2- Trp),
3.58 (1H, dd, JB, = 5.3 Hz, b-CH2- Trp), 3.06–2.94 (3H, m, b-CH2-
a-H-Asp), 4.23 (1H,
a
a
a
Asp, and -CH2OH- Trp), 2.73 (1H, dd, JA,B = 17.0, JB, = 6.5 Hz, b-
a
CH2- Asp), 2.45 (1H, br s, OH), 1.40 (9H, s, 3 ꢂ BOC). 13C NMR
(125 MHz, DMSO-d6): d 170.6 (2C, 2 ꢂ C@O), 155.5 (1C, C@O-
BOC), 136.4–111.3 (14C, Ar), 78.7 (1C, -OC(CH3)3- BOC), 65.9 (1C,
Trp), 7.05 (1H, d, J1,2 = 2.3 Hz, H2-Trp), 6.81 (1H, br d, JNH, = 7.7 Hz,
a
NH- Trp), 5.34 (1H, br d, JNH, = 7.7 Hz, NH Met), 5.10 (2H, br s, -
a
-OCH2- Bn), 62.1 (1C, -OCH2- Trp), 52.0 (1C, C
a
- Trp), 51.4 (1C,
OCH2- Bn), 4.79 (1H, m,
and -H-Trp), 4.27–4.17 (2H, m, H9-Fmoc and
(1H, dd, JA,B = 10.3, J ,A = 4.8 Hz, -CH2Br- Trp), 3.39 (1H, dd, J
a
-H- Asp), 4.48–4.37 (3H, m, CH2-Fmoc
Ca- Asp), 36.7 (1C, b-CH2- Asp), 28.4 (1C, -OC(CH3)3- BOC), 26.6
a
a-H-Met), 3.51
(1C, b-CH2- Trp). HRMS: Calcd for C27H33N3O6: m/z 496.2447.
a
Found: m/z 496.2458.
,B = 4.1 Hz, -CH2Br- Trp), 3.11–2.99 (3H, m, b-CH2- Trp and b-
a
Dipeptide BOC-Asp(OBn)-Tryptophanol (2.30 g, 4.64 mmol)
was dissolved in TFA (20 mL) and was left at rt for 10 min. The
CH2- Asp), 2.71 (1H, dd, JA,B = 17.2, J ,B = 6.4 Hz, Asp b-CH2- Asp),
2.45 (2H, br m, -SCH2- Met), 2.07 (4H, br s, b-CH2- Met, and
a

1422
B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
-SCH3- Met), 1.84 (1H, m, b-CH2- Met). MALDI-TOFMS: m/z 833.4
(M+Na).
as of a complicated pattern of overlapping rhamnose CH3 reso-
nances near d 1.3.
3.1.11. Fmoc-Met-Asp(OtBu)-tryptophanol 20
3.1.9. Protected glycopeptide 3
L
-Tryptophanol34 (3.77 g, 19.8 mmol), Fmoc-Asp(Ot-Bu)-OH
The trisaccharide thiol 4 (0.098 g, 0.13 mmol) and the tripeptide
bromide 5 (0.103 g, 0.127 g) were combined in EtOAc (15 mL) and
were stirred with satd aq NaHCO3 (2 mL). Bu4NHSO4 (0.032 g) was
added, and the mixture was stirred rapidly at rt for 5 h. The reac-
tion mixture was diluted with EtOAc (50 mL) and water (20 mL),
and the separated aq phase was further extracted with EtOAc
(30 mL). The combined extracts were dried over MgSO4 and were
concentrated to dryness. The residue was purified by flash chroma-
tography on silica gel (1:2 hexanes–EtOAc) to give the product as a
colorless solid (43 mg). Analysis by 1H NMR spectroscopy indicated
a purity of approximately 80%, along with one major, unidentified
contaminant that exhibited only peptide resonances. Further puri-
fication by preparative HPLC (C-18 reversed phase, 80:20–5:95
H2O–CH3CN over 30 min) gave the pure glycopeptide 3 as a color-
less solid (30 mg, 16%). 1H NMR (400 MHz, CD2Cl2): d 8.31 (1H, br s,
NH- Trp ring), 7.81–7.28 (15H, m, 13 Ar-[Fmoc and Bn], H4-, H7-
(8.09 g, 19.7 mmol), and HOBt (2.67 g, 19.7 mmol) were combined
in dry THF (70 mL). DCC (4.07 g, 19.7 mmol) was added, and the
mixture was stirred at rt for 1 h. After filtration to remove DCU,
the solvent was removed by rotary evaporation, and the residue
was partitioned between EtOAc (250 mL) and satd aq NaHCO3
(50 mL). The organic phase was washed with 10% citric acid
(50 mL), and then with satd aq NaHCO3 (2 ꢂ 50 mL) and satd aq
NaCl (50 mL). The solution was dried over MgSO4 and was evapo-
rated to an amorphous solid. The crude product was purified by
flash chromatography on silica gel (1:2 hexanes–EtOAc) to give
Fmoc-Asp(OtBu)-Tryptophanol as a colorless hard foam (9.11 g,
79%). 1H NMR (400 MHz, CDCl3): d 8.07 (1H, br s, NH-Trp ring),
7.82–7.22 (10H, m, H4-, H7-Trp, Ar-Fmoc), 7.16 (1H, br t,
J5,6 ꢄ J6,7 ꢄ 7.4 Hz, H6-Trp), 7.13 (1H, ddd, J4,5 = 7.9, J5,7 = 0.8 Hz,
H5-Trp), 7.06 (1H, d, J1,2 = 1.5 Hz, H2-Trp), 6.61 (1H, br d,
JNH, = 7.0 Hz, NH–Trp), 5.76 (1H, br d, JNH, = 8.4 Hz, NH–Asp),
a
a
Trp), 7.20 (1H, br d, J ,NH = 8.5 Hz, NH -Asp), 7.15 (1H, ddd,
a
4.47 (1H, br m,
a
-H- Asp), 4.40 (1H, dd, JA,B = 10.5, JA,9 = 7.2 Hz,
J6,7 = 8.1, J5,6 = 7.3, J4,6 = 1.0 Hz, H6-Trp), 7.08 (1H, ddd, J4,5 = 7.9,
J4,6 = 0.9 Hz, H5-Trp), 7.01 (1H, d, J1,2 = 2.2 Hz, H2-Trp), 6.72 (1H,
CH2-Fmoc), 4.33 (1H, dd, JB,9 = 7.0 Hz, CH2-Fmoc), 4.23 (1H, m,
H-Trp), 4.19 (1H, br t, J ꢄ 7.0 Hz, H9-Fmoc), 3.70 (1H, dd,
JA,B = 11.2, JA, = 3.4 Hz, -CH2OH- Trp), 3.61 (1H, dd, JB, = 5.6 Hz, -
a
br d, J ,NH = 8.0 Hz, NH-Trp), 5.38 (1H, br d, J ,NH = 7.1 Hz, NH-
a
a
a
a
Met), 5.29–5.20 (4H, m, H2-, H200-, H30-, H300-rha), 5.11–4.99 (6H,
CH2OH- Trp), 3.03 (1H, dd, JA,B = 17.4, JA, = 7.0 Hz, b-CH2- Trp),
a
m, -OCH2- Bn, H1-, H4-, H40-, H400-rha), 4.92 (1H, d, J1,2 = 1.6 Hz,
2.98 (1H, dd, JB, = 7.0 Hz, b-CH2- Trp), 2.84 (1H, dd, JA,B = 17.1,
a
H10-rha), 4.77(1H, d, J1,2 = 1.5 Hz, H100-rha), 4.70 (1H, m,
a
-H-
Asp), 4.47 (1H, dd, JA,B = 10.5, JA,9 = 6.7 Hz, CH2-Fmoc), 4.41–4.29
(2H, m, CH2-Fmoc, -H- Trp), 4.22 (1H, t, J = 6.7 Hz, H9-Fmoc),
4.17 (1H, m, -H- Met), 4.10 (1H, m, H5-rha), 4.02 (1H, dd,
J2,3 = 3.4, J3,4 = 9.8 Hz, H3-rha), 3.95 (1H, m, H5-rha), 3.89 (1H, dd,
JA, = 7.1 Hz, b-CH2- Asp), 2.61 (1H, dd, JB, = 6.8 Hz, b-CH2- Asp),
a
a
2.02 (1H, br s, OH), 1.41 (9H, s, tBu). 13C NMR (125 MHz, DMSO-
d6): d 170.5, 169.7 (2C, 2 ꢂ C@O), 156.1 (1C, C@O-Fmoc), 144.1–
111.4 (20C, Ar), 80.4 (1C, -OC(CH3)3–t-Bu), 66.1 (1C, -OCH2- Fmoc),
a
a
62.3 (1C, -OCH2- Trp), 52.1 (1C, Ca- Trp), 51.9 (1C, Ca- Asp), 46.9
J1 ,2 = 1.6, J2 3 = 3.4 Hz, H20-rha), 3.83 (1H, m, H5-rha), 3.04–2.92
(3H, m, b-CH2- Trp, and b-CH2- Asp), 2.80–2.69 (3H, m, -SCH2-
Trp, and b-CH2- Asp), 2.44 (2H, br t, J = 6.8 Hz, -SCH2- Met), 2.16
(3H, s, OAc), 2.12 (3H, s, CH3–OAc), 2.08 (3H, s, CH3–OAc), 2.06
(3H, s, -SCH3- Met), 2.07 (9H, s, 3 ꢂ OAc), 2.01 (1H, m, b-CH2-
Met), 1.98 (3H, s, OAc), 1.77 (1H, m, b-CH2- Met), 1.19 (3H, d,
J = 6.3 Hz, CH3-rha) 1.16 (3H, d, J = 6.3 Hz, CH3-rha) 1.13 (3H, d,
J = 6.2 Hz, CH3-rha). 13C NMR (100 MHz, CD2Cl2): d 171.9–169.8
(11C, 11 ꢂ C@O), 156.8 (1C, C@O-Fmoc), 144.2–111.2, (26C, Ar),
100.4 (1C, C100-rha), 99.9 (1C, C10-rha), 83.5 (1C, C1-rha), 77.8
(1C, C20-rha), 75.4 (1C, C30-rha), 73.3 (1C, C2-rha), 73.2 (1C, C30-
rha), 71.1 (2C, 2 ꢂ C4-rha), 70.5 (1C, C4-rha), 70.1 (1C, C200-rha),
69.1(1C, C300-rha), 67.8 (2C, 2 ꢂ C5-rha), 67.6 (1C, C5-rha), 67.5
0
0
0
0
(1C, C9-Fmoc), 38.2 (1C, b-CH2- Asp), 28.0 (1C, -OC(CH3)3- t-Bu),
26.6 (1C, b-CH2 -Trp). HRMS: Calcd for C34H37N3O6: m/z
584.2760. Found: m/z 584.2758.
The Fmoc-protected dipeptide Fmoc-Asp(OtBu)-Tryptophanol
(8.31 g, 14.2 mmol) was dissolved in dry DMF containing 20% v/v
piperidine (100 mL). After 10 min at rt, the solvents were removed
by rotary evaporation under high vacuum to give the free amino
compound H-Met-Asp(OtBu)-Tryptophanol as a crystalline solid
residue. This was dissolved in dry THF (100 mL) and Fmoc-Met-
OH (5.34 g, 14.3 mmol) was added, followed by HOBt (1.90 g,
14.1 mmol) and DCC (2.97 g, 14.3 mmol). The mixture was stirred
at rt for 15 h and was then filtered to remove DCU. The THF was
removed to leave a residue that was partitioned between EtOAc
(250 mL) and satd aq NaHCO3 (50 mL). The EtOAc layer was sepa-
rated and was washed with 10% citric acid (50 mL), satd aq NaH-
CO3 (2 ꢂ 50 mL), and satd aq NaCl (50 mL). The solution was
dried (MgSO4) and concentrated to give the crude tripeptide as a
pale-yellow solid. Purification by flash chromatography (EtOAc)
gave the protected tripeptide 20 as a white solid (8.65 g, 85%). 1H
NMR (500 MHz, CDCl3): d 8.12 (1H, br s, NH- Trp ring), 7.80–7.28
(11H, m, 8 ꢂ Ar Fmoc, H4-, H7-Trp, NH- Asp), 7.17 (1H, br t,
J5,6 ꢄ J6,7 ꢄ 7.5 Hz, H6-Trp), 7.10 (1H, br t, J5,6 ꢄ J4,5 ꢄ 7.5 Hz, H5-
(1C, CH2-Fmoc), 67.1 (1C, OCH2-Bn), 55.0 (1C, C
a-Met), 50.6 (1C,
Ca
-Trp), 50.0 (1C, C -Asp), 47.6 (1C, C9-Fmoc), 36.0 (1C, SCH2-
a
Trp), 35.7 (1C, Cb-Asp), 31.1 (1C, Cb-Met), 30.5 (1C, SCH2-Met),
29.4 (1C, Cb-Trp), 21.1–20.9 (7C, 7 ꢂ OAc) 17.7, 17.6, 17.4, (3C,
3 ꢂ C6-rha), 15.5 (1C, SCH3-Met). MALDI-TOFMS: m/z 1519.9
(M+Na).
3.1.10. Attempted deprotection of glycopeptide 3
The protected glycopeptide 3 (30 mg) was treated with 0.1 M
NaOMe in MeOH (4 mL) for 5 h at rt. The reaction mixture was
neutralized by addition of HOAc, filtered, and concentrated to a so-
lid residue. Flash chromatography on silica gel (4:2:1 EtOAc–
MeOH–H2O) and combining of those fractions that appeared as a
single spot on TLC gave a product free from protecting-group rem-
nants. However, analysis by HPLC showed this to be a mixture of
four closely eluting compounds with a ratio of 2:2:1:1. The MALDI
mass spectrum of the mixture showed a single, major mass corre-
sponding to loss of 18 mass units from the expected glycopeptide 1
(calcd for C38H58N4O16S2Na (M+Na) 913.31, found 895.15). The 1H
NMR spectrum in CD3OD also indicated a mixture of compounds,
as judged by the presence of several SCH3 singlets near d 2.0 as well
Trp), 7.04 (1H, br s, H2-Trp), 6.84 (1H, d, J ,NH = 8.0 Hz, NH-Trp),
a
5.57 (1H, d, J ,NH = 6.8 Hz, NH- Met), 4.70 (1H, m,
a
-H- Asp), 4.46
(1H, dd, JA,B = 10.7, JA,9 = 6.9 Hz, CH2-Fmoc), 4.42 (1H, dd,
JB,9 = 6.9 Hz, CH2-Fmoc), 4.28–4.18 (3H, m, -H- Met, -H- Trp,
H9-Fmoc), 3.70 (1H, dd, JA,B = 11.4, JA, = 3.4 Hz, -CH2OH- Trp),
a
a
a
a
3.60 (1H, dd, JB, = 5.3 Hz, -CH2OH-Trp), 3.02 (1H, dd, JA,B = 14.7,
a
J
a
,A = 7.5 Hz, b-CH2-Trp), 2.97 (1H, dd, J ,B = 6.4 Hz, b-CH2- Trp),
a
2.90 (1H, dd, JA,B = 16.8, J ,A = 4.3 Hz, b-CH2-Asp), 2.59 (1H, dd,
a
J
a
,B = 6.4 Hz, b-CH2-Asp), 2.48 (2H, m, SCH2- Met), 2.30 (1H, br s,
OH), 2.08 (3H, s, SCH3- Met), 2.05 (1H, m, b-CH2- Met), 1.86 (1H,
m, b-CH2–Met), 1.37 (9H, s, OtBu). 13C NMR (125 MHz, DMSO-
d6): d 171.4, 169.7 169.4 (3C, 3 ꢂ C@O), 156.1 (1C, C@O-Fmoc),

B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
1423
143.9–111.1 (20C, Ar), 80.5 (1C, OtBu), 65.7 (1C, CH2-Fmoc), 61.9
(1C, OCH2-Trp), 54.0 (1C, C -Met), 51.9 (1C, C -Trp), 49.7 (1C,
-Asp), 46.6 (1C, C9-Fmoc), 37.3 (1C, b-CH2–Asp), 31.5 (1C, b-
(3C, 3 ꢂ C6-rha), 15.5 (1C, -SCH3-Met). HRMS: Calcd for
C71H90N4O25S2: m/z 1463.5413. Found: m/z 1463.5393.
a
a
Ca
CH2–Met), 29.5 (1C, SCH2–Met), 27.6 (1C, OtBu), 26.3 (1C, b-CH2–
Trp), 14.6 (1C, SCH3–Met). HRMS: Calcd for C39H46N4O7S:
715.3165. Found: 715.3159.
3.1.13. Deprotection of glycopeptide 18
The partially purified glycopeptide 18 (>80% pure, 0.510 g,
ꢁ0.279 mmol) was dissolved in CH2Cl2 (20 mL). Tri-isopropylsilane
(0.3 mL) and TFA (6.0 mL) were added, and the mixture was kept at
rt for 2.5 h. The solvents were evaporated, and the residue was par-
titioned between CH2Cl2 (80 mL) and satd aq NaHCO3 (30 mL). The
emulsified organic phase was separated and was washed with 2 M
HCl (30 mL) and satd aq NaCl (2 ꢂ 30 mL). The solvents were re-
moved, and the residue was purified by flash chromatography
(10:1 to 4:1 EtOAc–MeOH) to give the purified, free b-carboxylic
acid-aspartate, glycopeptide. At this stage the impurity that was
present in the protected glycopeptide was easily separated as a fas-
ter running component and was characterized as the acetylated tri-
saccharide 1,10-disulfide 15 by NMR and MALDI spectra (data not
shown).
3.1.12. Glycopeptide 18
The protected tripeptide 20 (1.08 g, 1.51 mmol) was dissolved
in CH2Cl2 (30 mL) and was stirred at rt while triphenylphosphine
(0.550 g, 1.10 mmol) and CBr4 (0.709 g, 2.14 mmol) were added.
After 1 h, additional CH2Cl2 (50 mL) was added and the solution
was washed with satd aq NaHCO3 (20 mL), dried over MgSO4,
and concentrated. The residue was purified by flash chromatogra-
phy (1:2 hexanes–EtOAc) to give the tripeptide bromide 19 as a
pale-yellow foam (1.24 g). This material was only of limited stabil-
ity at rt and was used immediately in the next step.
The bromide (1.24 g, 1.59 mmol) was combined with the trisac-
charide thiol 4 (0.771 g, 1.01 mmol) in EtOAc (50 mL) and was stir-
red with satd aq NaHCO3 (20 mL). Bu4NHSO4 (0.223 g) was added,
and the mixture was stirred rapidly at rt for 6 h. The reaction mix-
ture was diluted with EtOAc (50 mL) and water (20 mL), and the
separated aq phase was further extracted with EtOAc (30 mL).
The combined extracts were washed with 10% citric acid (30 mL)
and satd aq NaHCO3 (2 ꢂ 30 mL), and were then dried over MgSO4
and concentrated to dryness. The residue was purified by flash
chromatography on silica gel (1:2–100% hexanes–EtOAc) to give
the product as a colorless, hard foam (1.01 g). Analysis by 1H
NMR spectroscopy indicated that this material was a mixture of
the expected glycopeptide 18 (>80%) along with a single impurity
that exhibited only typical rhamnose resonances. An analytical
sample was further purified by preparative HPLC (C-18 reversed
phase, 95:5–5:95 H2O–CH3CN over 40 min) to give first, an impu-
rity, a compound that was tentatively identified as the disulfide 15
resulting from oxidation of the trisaccharide thiol 4, and then, the
pure glycopeptide 18 as a pure colorless solid (16%). 1H NMR
(500 MHz, CD2Cl2): d 8.34 (1H, br s, NH-Trp ring), 7.82–7.30
The glycopeptide fraction was dissolved in 0.2 M NaOMe in
MeOH (12 mL) and was kept at rt for 6 h. The base was neutralized
by addition of HOAc and the volatile material was removed under
reduced pressure. Purification by flash chromatography (4:2:1
EtOAc–MeOH–H2O) gave the glycopeptide 1 as a colorless solid.
Analysis of the product by 1H NMR spectroscopy indicated the
presence of NaOAc (19 wt %, singlet at d 1.78). This led, after allow-
ing for this impurity, to an estimate of 32% for the yield of the
deprotection sequence. 1H NMR (500 MHz, D2O): d 7.69 (1H, d,
J4,5 = 7.5 Hz, H4-Trp), 7.47 (1H, d, J6,7 = 8.0 Hz, H7-Trp), 7.24 (1H,
br s, H2-Trp), 7.21 (1H, br t, J5,6 ꢄ J6,7 ꢄ 8.0 Hz, H6-Trp), 7.14 (1H,
br t, H5-Trp), 5.13 (2H, br s, 2 ꢂ H1-rha), 4.93 (1H, d, J1,2 = 1.5 Hz,
H1-rha), 4.63 (1H, dd,
a-H Asp), 4.26 (1H, m, a-H Trp), 4.08–4.01
(4H, m, 3 ꢂ H2-rha, -H Met), 3.96 (1H, m, H5-rha), 3.89 (1H, dd,
a
J2,3 = 3.4, J3,4 = 9.9 Hz, H3-rha), 3.76 (1H, dd, J2,3 = 3.4, J3,4 = 9.8 Hz,
H3-rha), 3.73 (1H, m, H5-rha), 3.69 (1H, dd, J2,3 = 3.3, J3,4 = 9.7 Hz,
H3-rha), 3.67 (1H, m, H5-rha), 3.51(t, 1H, J = 9.6, H4-rha),
3.45(1H, t, J = 9.7 Hz, H4-rha), 3.41(1H, t, J = 9.7 Hz, H4-rha), 3.09
(1H, dd, JA,B = 14.7 Hz, JA, = 5.8 Hz, b-CH2 Trp), 2.96 (1H, dd,
a
(10H, m, 8ꢂ Ar-Fmoc, H4-, H7-Trp), 7.26 (1H, br d, J ,NH = 8.3 Hz,
JB, = 7.3 Hz, b-CH2 Trp), 2.92 (1H, dd, JA,B = 13.7, JA, = 4.3 Hz, -
a
a
a
NH-Asp), 7.16 (1H, br t, J5,6 ꢄ J6,7 ꢄ 7.5 Hz, H6-Trp), 7.09 (1H, br t,
J5,6 ꢄ J4,5 ꢄ 7.5 Hz, H5-Trp), 7.05 (1H, d, J1,2 = 2.1 Hz, H2-Trp), 6.77
SCH2 Trp), 2.79 (1H, dd, JB, = 9.2 Hz, -SCH2 Trp), 2.63 (1H, dd,
a
JA,B = 16.0, J ,A = 4.6 Hz, b-CH2 Asp), 2.49 (1H, dd, J ,B = 9.9 Hz, b-
a
a
(1H, br d, J ,NH = 8.2 Hz, NH- Trp), 5.50 (1H, br d, J ,NH = 7.2 Hz,
CH2 Asp), 2.44 (1H, dt, JA,B = 13.5, J b,A = 7.4 Hz, -SCH2 Met), 2.33
(1H, dt, J b,B = 7.2 Hz, -SCH2 Met), 2.02 (2H, m, b-CH2 Met), 1.96
(3H, s, -SCH3 Met), 1.25 (3H, d, J = 6.3 Hz, CH3- rha) 1.23 (3H, d,
J = 6.4 Hz, CH3-rha) 1.22 (3H, d, J = 6.3 Hz, CH3-rha). 13C NMR
(125 MHz, D2O): d 177.3, 172.2, 168.7 (each 1C, 3 ꢂ C@O), 136.2,
127.3, 124.4, 121.9, 119.4, 118.7, 112.0, 110.3 (each, 1C, 8 ꢂ Ar
Trp), 102.4 (1C, C100-rha), 100.9 (1C, C10-rha), 86.6 (1C, C1-rha),
78.2 (1C, C20-rha), 77.8 (1C, C3-rha), 72.2, 72.1, 71.9 (each 1C,
3 ꢂ C4-rha), 70.2, 70.1, 70.0 (each 1C, 2 ꢂ C3-, C2-rha), 69.5, 69.4,
a
a
NH- Met), 5.30–5.23 (3H, m, H2-, H200-, H300-rha), 5.22 (1H, br s,
H1-rha), 5.11–4.99 (4H, m, H-30, H4-, H40-, H400-rha), 4.93 (1H, d,
J1,2 = 1.5 Hz, H10-rha), 4.78 (1H, d, J1,2 = 1.4 Hz, H100-rha), 4.65 (1H,
m,
4.41 - 4.33 (2H, m, CH2-Fmoc,
Fmoc,
a
-H-Asp), 4.47 (1H, dd, JA,B = 10.5, JA,9 = 7.1 Hz, CH2-Fmoc),
a
-H-Trp), 4.26–4.19 (2H, m, H9-
a-H-Met), 4.11 (1H, m, H5-rha), 4.03 (1H, dd, J2,3 = 3.4,
J3,4 = 9.8 Hz, H3-rha), 3.96 (1H, m, H5-rha), 3.90 (1H, br m, H20-
rha), 3.84 (1H, m, H5-rha), 3.02 (2H, d, J = 6.3 Hz, b-CH2- Trp),
2.83 (1H, dd, JA,B = 16.8, J ,A = 4.8 Hz, b-CH2- Asp), 2.78 (2H, d,
69.3 (each 1C, 3 ꢂ C5-rha), 52.3 (1C, C
a Met), 52.0 (1C, Ca Asp),
51.3 (1 C, C Trp), 39.4 (1C, b-CH2 Asp), 36.1 (1C, -SCH2Trp), 30.0
a
J = 6.4 Hz, -SCH2- Trp), 2.60 (1H, dd, J ,B = 6.1 Hz, b-CH2- Asp),
a
a
2.49 (2H, br t, J = 6.8 Hz, -SCH2- Met), 2.17 (3H, s, OAc), 2.13 (3H,
s, CH3-OAc), 2.10 (3H, s, CH3-OAc), 2.08 (3H, s, -SCH3- Met), 2.05
(1H, m, b-CH2- Met), 2.04 (9H, s, 3 ꢂ OAc), 1.99 (3H, s, OAc), 1.85
(1H, m, b-CH2- Met), 1.41 (9H, s, Ot-Bu), 1.21 (3H, d, J = 6.3 Hz,
CH3-rha) 1.18 (3H, d, J = 6.3 Hz, CH3-rha) 1.14 (3H, d, J = 6.2 Hz,
(1C, b-CH2 Met), 29.4 (1C, b-CH2 Trp), 28.2 (1C, -SCH2 Met), 16.8,
(2C, 2 ꢂ C6-rha), 16.7 (1C, C6-rha), 14.2 (1C, -SCH3 Met). MALDI-
TOFMS: Calcd for C38H58N4O16S2Na (M+Na) m/z 913.31. Found:
m/z 913.11.
CH3-rha). 13C NMR (100 MHz, CD2Cl2):
d
171.3–169.7 (11C,
3.1.14. Bromo compound 21
11 ꢂ C@O), 156.5 (1C, C@O-Fmoc), 144.0–111.2, (20C, Ar), 100.2
(1C, C100-rha), 99.8 (1C, C10-rha), 83.4 (1C, C1-rha), 77.7 (1C, C20-
rha), 75.3 (1C, C30-rha), 73.2 (1C, C2-rha), 73.1 (1C, C30-rha), 71.0
(2C, 2 ꢂ C4-rha), 70.5 (1C, C4-rha), 70.1 (1C, C200-rha), 69.0(1C,
C300-rha), 67.8 (1C, C5-rha), 67.7 (2C, 2 ꢂ C5-rha), 67.6 (1C, CH2-
To a stirred mixture of L
-tryptophanol34 (3.04 g, 16 mmol) and
NaHCO3 (1.34 g) in water and acetone (50 mL, 1:1) was added
Fmoc-OSu (5.3 g, 16 mmol). After 5 min, a thick white precipitate
formed, and the reaction mixture was stirred at rt for 1 h. The solid
was then collected by filtration, and was washed with hot H2O
(100 mL) and hot Et2O (100 mL) to afford Fmoc-tryptophanol
(6.3 g, 95%) as a white solid. 1H NMR (500 MHz, acetone-d6): d
10.02 (1H, br s, NH-Trp ring), 7.85 (2H, d, J = 7.6 Hz, H4-, H5-Fmoc),
7.72 (1H, d, J = 7.7 Hz, H4-Trp), 7.68 (2H, d, J = 6.9 Hz, H1-,
Fmoc), 54.9 (1C, Ca- Met), 50.5 (1C, Ca- Trp), 49.9 (1C, Ca- Asp),
47.5 (1C, C9-Fmoc), 36.7 (1C, b-CH2- Asp), 35.9 (1C, -SCH2- Trp),
31.3 (1C, b-CH2- Met), 30.5 (1C, -SCH2- Met), 29.4 (1C, b-CH2-
Trp), 28.1 (1C, OtBu), 21.1–20.8 (7C, 7 ꢂ OAc) 17.7, 17.6, 17.4,

1424
B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
H8-Fmoc), 7.41 (2H, d, H3-, H6-Fmoc), 7.36 (1H, d, J = 7.8 Hz, H7-
Trp), 7.30 (2H, dd, H2-, H7-Fmoc), 7.20 (1H, br s, H2-Trp), 7.09
(1H, br t, J = 7.3 Hz, H6-Trp), 7.02 (1H, br t, J = 7.3 Hz, H5-Trp),
6.31 (1H, d, J = 8.4 Hz, NH-Trp), 4.34 (1H, dd, J = 11.0, 7.3 Hz, CH2-
Fmoc), 4.27 (1H, dd, CH2-Fmoc), 4.20 (1H, dd, H9-Fmoc), 4.08–
mixture was stirred vigorously at rt for 12 h. It was then diluted
with EtOAc and was washed successively with satd aq NaHCO3
and brine. The organic layer was dried over Na2SO4, concentrated,
dissolved in CH2Cl2, and purified by flash chromatography (1:1
hexanes–EtOAc) to afford the glycopeptide 22 (102 mg, 92%) as a
white crystalline solid. 1H NMR (500 MHz, CD2Cl2): d 8.35 (1H, br
s, NH- Trp ring), 7.80 (2H, d, J = 7.55 Hz, H4-, H5-Fmoc), 7.68 (1H,
d, J = 7.78 Hz, H4-Trp), 7.64–7.57 (2H, m, H1-, H8-Fmoc), 7.47–
7.38 (3H, m, H3-, H6-Fmoc, H4-Trp), 7.36–7.30 (2H, m, H2-, H7-
Fmoc), 7. 23–7.18 (1H, m, H6-Trp), 7.17–7.10 ((1H, m, H5-Trp),
7.07 (1H, br s, H2-Trp), 5.37–5.33 (2H, m, H2-rha, NH-Dmab),
5.32–5.28 (2H, m, NH-Trp, H300-rha), 5.27 (1H, dd, J2”,3” = 3.52,
J2”,1” = 1.82 Hz, H2”-rha), 5.22 (1H, s, H1-rha), 5.12–5.06 (2H, m,
H4-, H4”-rha), 5.05–5.03 (2H, m, H30-, H40-rha), 4.95 (1H, d,
3.96 (1H, m,
a-H-Trp), 3.68–3.58 (2H, m, CH2OH-Trp), 3.09 (1H,
dd, J = 13.9, 6.9 Hz, b-CH2-Trp), 3.00 (1H, dd, J = 7.3 Hz, b-CH2-
Trp), 2.89 (1H, br s, CH2OH-Trp). 13C NMR (150 MHz, acetone-d6):
d 156.3 (1C, C@O-urethane), 144.5 (2C, C8a-, C9a-Fmoc), 141.4
(2C, C4a-, C4b-Fmoc), 137.0 (1C, C3a-Trp), 128.3 (1C, C7a-Trp),
127.8 (2C, C3-, C6-Fmoc), 127.2 (2C, C2-, C7-Fmoc), 125.5 (2C,
C1-, C8-Fmoc), 123.4 (1C, C2-Trp), 121.4 (1C, C5-Trp), 120.1 (2C,
C4-, C5-Fmoc), 118.9 (1C, C4-Trp), 118.8 (1C, C6-Trp), 112.1 (1C,
C3-Trp), 111.4 (1C, C7-Trp), 66.1 (1C, CH2-Fmoc), 63.5 (1C, CH2OH),
54.2 (1C, Ca-Trp), 47.6 (1C, C9-Fmoc), 27.1 (1C, bCH2-Trp). MALDI-
J1 ,2 = 1.68 Hz, H10-rha), 4.81 (1H, d, J1”,2” = 1.53 Hz, H1”-rha),
4.45–4.36 (2H, m, CH2-Fmoc), 4.28–4.14 (3H, m, -H-Trp, H9-
0
0
TOFMS: m/z 435.7 (M+Na), 413.4 (M+H). Anal. Calcd for
C26H24N2O3: C, 75.71; H, 5.86; N 6.79. Found: C, 76.02; H, 5.93; N
6.54.
a
Fmoc, H5-rha), 4.07 (1H, dd, J3,4 = 9.78 Hz, J3,2 = 3.48 Hz, H3-rha),
4.02–3.95 (1H, m, H5”-rha), 3.94–3.91 (1H, m, H20-rha), 3. 90–
3.83 (1H, m, H50-rha), 3.07 (2H, br t, b -CH2-Trp) 2.85 (2H, br t,
CH2S), 2.22–1.98 (21H, 7 ꢂ OAc), 1.34–1.15 (9H, m, 3 ꢂ CH3-rha).
13C NMR (150 MHz, CD2Cl2): d 170.5–169.9 (7C, 7 ꢂ OAc), 155.9
(1C, C@O-urethane), 144.3 (2C, C8a-, C9a-Fmoc), 141.5 (2C, C4a-,
C4b-Fmoc), 136.5 (1C, C3a-Trp), 127.8 (1C, C7a-Trp), 127.8 (3C,
C3-, C6-Fmoc), 127.2 (2C, C2-, C7-Fmoc), 125.3 (2C, C1-, C8-Fmoc),
123.2 (1 C, C2-Trp), 122.3 (1 C, C5-Trp), 120.1 (2 C, C4-, C5-Fmoc),
119.7 (1 C, C6-Trp), 118.9 (1C, C7-Trp), 111.4 (1C, C4-Trp), 111.4
(1C, C3-Trp), 100.3 (1C, C1”-rha), 99.8 (1C, C10-rha), 83.9 (1C, C1-
rha), 77.5 (1C, C20-rha), 75.1 (1C, C3-rha), 73.1 (1C, C2-rha), 72.9
(1C, C4”-rha), 70.9 (1C, C30-rha), 70.8 (1C, C4-rha), 70.3 (1C, C40-
rha), 69.9 (1C, C2”-rha), 68.9 (1C, C3”-rha), 67.8 (1C, C50-rha),
67.6 (1C, C5-rha), 67.4 (1C, C5”-rha), 66.6 (1C, CH2-Fmoc), 51.5
Method 1: To a stirred mixture of Fmoc-tryptophanol (0.98 g,
2.4 mmol) and CBr4 (1.34 g, 4 mmol) in dry CH2Cl2 at 0 °C was
added PPh3 (1.25 g, 4.8 mmol) in portions. The reaction mixture
was stirred at 0 °C for 2 h and was then concentrated under re-
duced pressure at rt. The residue was then purified by flash chro-
matography (3:1 hexanes–EtOAc) to afford the bromide 21 as a
pale-yellow powder (0.79 g, 65%). The spectral data were the same
as those for the compound obtained with method 2 (see below).
Method 2: To a stirred solution of Fmoc-tryptophanol (3 g,
7.3 mmol) in pyridine (150 mL) at 0 °C under a N2 atmosphere
was added MsCl (0.7 mL, 11 mmol). The reaction mixture was stir-
red at 0 °C for 1 h and at rt for 4 h. The reaction mixture was then
quenched with ice, and the solvent was evaporated. The residue
was then dissolved in EtOAc (500 mL), washed with water
(300 mL) and satd aq NaHCO3 (300 mL), and dried over Na2SO4,
and the solvent was evaporated. The crude material containing LiBr
(5.1 g, 58.4 mmol) in THF (75 mL) was refluxed under a N2 atmo-
sphere for 4 h. The solvent was then evaporated, and the mixture
was diluted with EtOAc (500 mL), washed with water
(5 ꢂ 300 mL) and brine (200 mL), dried over Na2SO4, and the sol-
vent was evaporated. The residue was then purified by column
chromatography (3:1 hexanes–EtOAc) to give the bromide 21
(3.3 g, 95% over the two steps), as a pale-yellow solid. 1H NMR
(500 MHz, acetone-d6): d 10.1 (1H, br s, NH-Trp ring), 7.87 (2H,
d, J = 7.8 Hz, H4-, H5-Fmoc), 7.68 (1H, d, J = 6.3 Hz, H4-Trp), 7.67
(2H, d, J = 7.3 Hz, H1-, H8-Fmoc), 7.43–7.38 (2H, m, H3-, H6-Fmoc,
H7-Trp), 7.31 (2H, dd, H2-, H7-Fmoc), 7.26 (1H, br s, H2-Trp), 7.12–
7.07 (1H, br t, J = 7.3 Hz, H6-Trp), 7.04 (1H, br t, J = 7.3 Hz, H5-Trp),
6.65 (1H, d, J = 7.8 Hz, NH-Trp), 4.34 (2H, d, J = 7.3 Hz, CH2-Fmoc),
(1C, Ca-Trp), 47.5 (1C, C9-Fmoc), 36.9 (1C, CH2S), 29.6(1C, b-CH2-
Trp), 21.1–20.6 (7C, 7 ꢂ OAc), 17.6–17.2 (3C, 3 ꢂ CH3-rha). MAL-
DI-TOFMS: m/z 1183.9 (M+Na), 1199.9 (M+K), 1160.1 (M+). Anal.
Calcd for C58H68N2O21S: C, 59.99; H, 5.90; N, 2.41. Found: C,
59.70; H, 6.06; N, 2.37.
3.1.16. Fmoc-Asp(ODmab)-OtBu (26)
To a mixture of Fmoc-Asp-OtBu (1.0 g, 2.4 mmol), Dmab-OH
(1.6 g, 4.9 mmol), DTBMP (0.5 g, 2.4 mmol), and HOBt (0.6 g,
3.6 mmol) in dry THF (20 ml), DIC (0.37 mL, 2.4 mmol) was added
dropwise. The reaction mixture was stirred for 12 h at rt, concen-
trated to dryness under vacuum, dissolved in CH2Cl2, and purified
by flash chromatography (2:1 hexanes–EtOAc) to afford the prod-
uct 26 as a white solid (1.25 g, 71%) after lyophilization in dioxane.
1H NMR (500 MHz, CD3OD): d 7.75 (2H, d, J = 7.5 Hz, H4-, H5-
Fmoc), 7.66–7.59 (2H, m, H1-,H8-Fmoc), 7.41 (2H, d, J = 8.0 Hz,
H-Dmab), 7.35 (3H, t, J = 7.5 Hz, H3-, H6-Fmoc), 7.26 (2H, t,
J = 7.4 Hz, H2-, H7-Fmoc), 7.09 (2H, d, J = 8.0 Hz, Ar-Dmab), 4.52
4.24–4.15 (2H, m,
a-H-Trp, H9-Fmoc), 3.69 (1H, dd, J = 10.3,
J = 4.88 Hz, CH2Br-Trp), 3.69 (1H, dd, J = 10.3 and 4.88 Hz, CH2Br-
Trp), 3.60 (1H, dd, J = 5.86 Hz, b-CH2-Trp), 3.19–3.08 (1H, m, b-
CH2-Trp). 13C NMR (150 MHz, acetone-d6): d 156.0 (1C, C@O-ure-
thane), 144.4 (2C, C8a-, C9a-Fmoc), 141.4 (2C, C4a-, C4b-Fmoc),
137.0 (1C, C3a-Trp), 127.9 (1C, C7a-Trp), 127.8 (2C, C3-, C6-Fmoc),
127.2 (2C, C2-, C7-Fmoc), 125.5 (2C, C1-, C8-Fmoc), 123.7 (1C, C2-
Trp), 121.6 (1C, C6-Trp), 120.1 (2C, C4-, C5-Fmoc), 119.0 (1C, C5-
Trp), 118.6 (1C, C4-Trp), 111.6 (1C, C7-Trp), 110.9 (1C, C3-Trp),
(1H, t, J = 6.4 Hz,
aH-Asp), 4.33 (1H, dd, J = 11.2 and 7.0 Hz, CH2-
Fmoc), 4.25 (1H, dd, J = 11.2, 7.3 Hz, CH2-Fmoc), 4.16 (1H, t,
J = 6.9 Hz, H9-Fmoc), 3.04–2.89 (3H, m, CH2CH-Dmab, bꢀCH2-
Asp), 2.83 (1H, dd, J = 16.1 and 7.3 Hz, bꢀCH2-Asp), 2.45–2.39
(4H, m, 2 ꢂ CH2 ring-Dmab), 2.01–1.99 (4H, m, 2 ꢂ CH2 ring-
Dmab), 1.82–1.69 (1H, m, CH2CH-Dmab), 1.41 (9H, s, OtBu), 1.04
(6H, s, 2 ꢂ CH3 ring-Dmab), 0.74–0.67 (6H, m, CH2CH(CH3)2-
Dmab). 13C NMR (150 MHz, CD3OD): d 176.7 (2C, C@O ring-Dmab),
171.7 (1C, C@CN Dmab), 170.5 (1C, COODmab), 170.2 (1C, COOtBu),
157. 0 (1C, C@O-urethane), 144.0 (2C, C8a-, C9a-Fmoc), 143.9 (1C,
CAr-N Dmab), 141.4 (2C, C4a-, C4b-Fmoc), 136.3 (1C, CAr-CH2O
Dmab), 129.0 (2C, CAr-Dmab), 127.7 (2C, C3-, C6-Fmoc), 127.0
(2C, C2-, C7-Fmoc), 126.5 (2C, CAr-Dmab), 125.1 (2C, C1-, C8-Fmoc),
119.9 (2C, C4-, C5-Fmoc), 107.4 (1C, C@CN), 82.1 (1C, COOC(CH3)3-
OtBu), 66.9 (1C, CH2-Fmoc), 65.5 (1C, CAr-CH2O-Dmab), 52.4 (2C,
66.2 (1C, CH2-Fmoc), 53.1 (1C, Ca-Trp), 47.4 (1C, C9-Fmoc), 36.9
(1C, CH2Br), 28.7 (1C, bCH2-Trp). MALDI-TOFMS: m/z 498.23,
497.3 (M+Na), 476.8, 475.3 (M+H). Anal. Calcd for C26H23BrN2O2:
C, 65.69; H, 4.88; N, 5.89. Found: C, 66.02; H, 5.04; N 5.59.
3.1.15. Glycopeptide 22
To a stirred mixture of the bromide 21 (46.4 mg, 98
trisaccharide glycosyl thiol 4 (75 mg, 98 mol) in EtOAc (3 mL),
satd aq NaHCO3 (3 mL), followed by tetrabutylammonium hydro-
gen sulfate (TBAHS) (133 mg, 39.2 mol) was added. The reaction
lmol) and
l
2 ꢂ CH2 ring-Dmab), 51.5 (1C, C
a-Asp), 47.1 (1C, C9-Fmoc), 38.2
(1C, CH2CH-Dmab), 36.4 (1C, bCH2-Asp), 29.8 (1C, CH2CH-Dmab),
l

B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
1425
27.1 (2C, 2 ꢂ CH3 ring-Dmab), 27.3 (3C, OtBu), 21.8 (2C,
CH2CH(CH3)2-Dmab). MALDI-TOFMS: m/z 745.2 (M+Na), 722.7
(M+H). Anal. Calcd for C43H50N2O8: C, 71.45; H, 6.97; N, 3.88.
Found: C, 71.14; H, 7.24; N, 3.62.
(5H, m, CH2-Fmoc,
a
-H-Fsp,
a
-H-Vet, H9-Fmoc), 3.06–2.84 (4H,
m, CH2CH-Dmab, b-CH2-Asp), 2.60–2.46 (2H, m, d-CH2-Met),
2.42 (2H, s, CH2 ring-Dmab), 2.06 (2H, s, CH2 ring-Dmab), 1.99
(3H, s, SCH3-Met), 1.95–1.83 (2H, m, bCH2-Met), 1.81–1.71 (1H,
m, CH2CH-Dmab), 1.05 (6H, s, 2 ꢂ CH3 ring-Dmab), 0.71 (6H, d,
J = 6.6 Hz, CH2CH(CH3)2-Dmab). 13C NMR (150 MHz, CD3OD): d
176.7 (2C, C@O ring-Dmab), 172.95 (1C, CO-amide), 171.7 (1C,
COODmab), 170.5 (1C, C@CN Dmab), 157.2 (1C, C@O-urethane),
144.1 (2C, C8a-, C9a-Fmoc), 143.9 (1C, CAr-N-Dmab), 141.4 (2C,
C4a-, C4b-Fmoc), 136.3 (1C, CAr-CH2O-Dmab), 128.9 (2C, CAr
Dmab), 127.6 (2C, C3-, C6-Fmoc), 127.0 (2C, C2-, C7-Fmoc),
126.5 (2C, CAr-Dmab), 125.1 (2C, C1-, C8-Fmoc), 119.8 (2C, C4-,
C5-Fmoc), 107.4 (1C, C@CN), 66.9 (1C, CH2-Fmoc), 65.5 (1C, CAr
3.1.17. Fmoc-Asp(ODmab)-OH (27)
Fmoc-Asp(ODmab)-OtBu (26) (1.0 g, 1.4 mmol) was dissolved in
a mixture of 1:1 CH2Cl2–TFA (10 mL), and the reaction mixture was
stirred for 1 h. The volatiles were then removed under vacuum, and
the residue was co-evaporated five times with toluene, dissolved in
CH2Cl2, and purified by flash chromatography (97:3 CH2Cl2–MeOH)
to give the product 27 as a pale-yellow solid (0.9 g, 97%). 1H NMR
(500 MHz, CD3OD): d 7.77 (2H, d, J = 7.5 Hz, H4-, H5-Fmoc), 7.69–
7.61 (2H, m, H1-, H8-Fmoc), 7.44 (2H, d, J = 8.1 Hz, ArH-Dmab),
7.37 (3H, t, J = 7.4 Hz, H3-, H6-Fmoc), 7.28 (2H, t, J = 7.4 Hz, H2-,
H7-Fmoc), 7.08 (2H, d, J = 8.1 Hz, ArH-Dmab), 5.17 (2H, d,
-
-
CH2O-Dmab), 54.8 (1C, C -Met), 54.3 (1C, C -Asp), 52.4 (2C,
a
a
2 ꢂ CH2 ring-Dmab), 47.2 (1C, C9-Fmoc), 39.0 (1C, CH2CH-Dmab),
36.2 (1C, Cb-Asp), 31.3 (1C, b-CH2-Met), 29.9 (1C, Cd-Met), 29.4
(1C, CH2CH-Dmab), 27.2 (2C, 2 ꢂ CH3 ring-Dmab), 21.7 (2C,
CH2CH(CH3)2-Dmab), 19.8 (1C, SCH3-Met. MALDI-TOFMS: m/z
706.4 (M+K), 690.1 (M+Na), 667.9 (M+H). Anal. Calcd for
C44H51N3O9S: C, 66.23; H, 6.44; N, 5.27. Found: C, 66.07; H,
6.31; N, 5.36.
J = 3.5 Hz, Ar-CH2O-Dmab), 4.52 (1H, t, J = 6.4 Hz,
aꢀH-Asp), 4.33
(1H, dd, J = 11.2, 7.0 Hz, CH2-Fmoc), 4.25 (1H, dd, J = 11.2, 7.3 Hz,
CH2-Fmoc), 4.16 (1H, t, J = 6.9 Hz, H9-Fmoc), 3.04–2.89 (3H, m,
CH2CH-Dmab, bꢀCH2-Asp), 2.83 (1H, dd, J = 16.1, 7.3 Hz, bꢀCH2-
Asp), 2.45–2.39 (4H, m, 2 ꢂ CH2 ring-Dmab), 2.01–1.99 (4H, m,
2 ꢂ CH2 ring-Dmab), 1.82–1.69 (1H, m, CH2CH-Dmab), 1.41 (9H, s,
OtBu), 1.04 (6H, s, 2 ꢂ CH3 ring-Dmab), 0.74–0.67 (6H, m,
CH2CH(CH3)2-Dmab). 13C NMR (150 MHz, CD3OD): d 176.7 (2C,
C@O ring-Dmab), 171.7 (1C, COODmab), 170.5 (1C, C@CN-Dmab),
170.2 (1C, COOtBu), 157. 0 (1C, C@O-urethane), 144.0 (2C, C8a-,
C9a-Fmoc), 143.9 (1C, CAr-N Dmab), 141.4 (2C, C4a-, C4b-Fmoc),
136.3 (1C, CAr-CH2ODmab), 129.0 (2C, CAr-Dmab), 127.7 (2C, C3-,
C6-Fmoc), 127.0 (2C, C2-, C7-Fmoc), 126.5 (2C, CAr-Dmab), 125.1
(2C, C1-, C8-Fmoc), 119.9 (2C, C4-, C5-Fmoc), 107.4 (1C, C@CN),
3.1.19. Fmoc-Met-Asp(ODmab)-Opfp (24)
Fmoc-Met-Asp(ODmab)-OH (28) (0.3 g, 0.4 mmol) and penta-
fluorophenol (0.1 g, 0.56 mmol) were dissolved in dry CH2Cl2
(7 mL), DIC (58 lL, 0.4 mmol) was added dropwise, and the reac-
tion mixture was stirred for 3 h at rt. The mixture was then cooled
on ice for 1 h and the diisopropylurea was filtered. The filtrate was
concentrated, and the crude product was purified by flash chroma-
tography (3:1?2:1 hexanes–EtOAc) to afford the activated dipep-
tide 24 as a white, crystalline solid (0.26 g, 73%). This material was
only of limited stability at rt and was generally used immediately
in the next step. 1H NMR (500 MHz, CD3OD): d 7.78 (2H, d,
J = 7.5 Hz, H4-, H5-Fmoc), 7.68–7.61 (2H, m, H1-, H8-Fmoc),
7.42–7.32 (4H, m, ArH-Dmab, H3-, H6-Fmoc), 7.28 (2H, t,
J = 7.4 Hz, H2-, H7-Fmoc), 7.11 (2H, d, J = 7.8 Hz, Ar-Dmab), 5.09
82.1 (1C, COOC(CH3)3-OtBu), 66.9 (1C, CH2-Fmoc), 65.5 (1C, CAr
-
CH2O-Dmab), 52.4 (2C, 2 ꢂ CH2 ring-Dmab), 51.5 (1C, C
a
-Asp),
47.1 (1C, C9-Fmoc), 38.2 (1C, CH2CH-Dmab), 36.4 (1C, bꢀCH2-
Asp), 29.8 (1C, CH2CH-Dmab), 27.1 (2C, 2 ꢂ CH3 ring-Dmab), 27.3
(3C, OtBu), 21.8 (2C, CH2CH(CH3)2-Dmab). MALDI-TOFMS: m/z
945.2 (M+Na), 722.7 (M+H). Anal. Calcd for C43H50N2O8: C, 71.45;
H, 6.97; N, 3.88. Found: C, 71.14; H, 7.24; N, 3.62.
(2H, s, Ar-CH2O-Dmab), 4.40–4.14 (5H, m, CH2-Fmoc, a-H-Fsp, a-
H-Vet, H9-Fmoc), 3.06–2.84 (4H, m, CH2CH-Dmab, b-CH2-Asp),
2.60–2.46 (2H, m, d-CH2-Met), 2.42 (2H, s, CH2 ring-Dmab), 2.06
(2H, s, CH2 ring-Dmab), 1.99 (3H, s, SCH3-Met), 1.95–1.83 (2H, m,
b-CH2-Met), 1.81–1.71 (1H, m, CH2CH-Dmab), 1.05 (6H, s,
2 ꢂ CH3 ring-Dmab), 0.71 (6H, d, J = 6.6 Hz, CH2CH(CH3)2-Dmab).
13C NMR (150 MHz, CD3OD): d 176.7 (2C, C@O ring-Dmab), 173.3
(1C, COOPfp), 170.1 (1C, CO-amide), 169.9 (1C, COODmab), 167.1
(1C, C@CN-Dmab), 157.2 (1C, C@O-urethane), 144.2 (2C, C8a-,
C9a-Fmoc), 143.9 (1C, CAr-N-Dmab), 142.1 (1C, CAr-OPfp), 141.4–
3.1.18. Fmoc-Met-Asp(ODmab)-OH (28)
2-Chlorotrityl resin (0.3 g, loading: 1.69 mmol) was swelled in
CH2Cl2 (10 mL) for 5 min. The resin was then washed with DMF
(10 mL). DIPEA (3 mL) was added, followed by the addition of
Fmoc-Asp(ODmab)-OH 27 (0.5 g, 0.75 mmol) dissolved in DMF
(4 mL). The flask was shaken for 1 h and the resin was washed
with DMF (3 ꢂ 10 mL) and CH2Cl2 (10 mL). A mixture of 80:16:4
CH2Cl2–MeOH–DIPEA (10 mL) was then introduced, and the flask
was shaken for 15 min. The operation was repeated once. The re-
sin was then washed with DMF (3 ꢂ 10 mL), and 25% piperidine in
DMF (10 mL) was added, and the flask was shaken for 10 min. The
operation was repeated for 30 min. The resin was washed with
DMF (3 ꢂ 10 mL), and Fmoc-Met-OH (0.57 g, 1.5 mmol), HBTU
(0.54 g, 1.4 mmol), HOBt (0.22 g, 1.4 mmol), and DIPEA (3 mL) dis-
solved in DMF (7 mL) were added. The flask was then shaken for
1 h. The resin was washed with DMF (3 ꢂ 10 mL) and CH2Cl2
(3 ꢂ 10 mL). TFA (5% in CH2Cl2, 10 mL) was added to the resin
and the flask was shaken for 1 h. The solution was collected in a
round-bottomed flask and the resin was washed with CH2Cl2
(3 ꢂ 5 mL) and MeOH (3 ꢂ 5 mL). The solvent was then removed
under vacuum, and the residue was azeotroped three times with
toluene, dissolved in CH2Cl2, and purified by flash chromatogra-
phy (1:1 hexanes–EtOAc) to afford the protected dipeptide 28 as
a yellow powder (0.4 g, 80%) after lyophilization in dioxane. 1H
NMR (500 MHz, CD3OD): d 7.76 (2H, d, J = 7.5 Hz, H4-, H5-Fmoc),
7.67–7.59 (2H, m, H1-, H8-Fmoc), 7.42–7.32 (4H, m, Dmab-ArH,
H3-, H6-Fmoc), 7.28 (2H, t, J = 7.4 Hz, H2-, H7-Fmoc), 7.11 (2H,
d, J = 7.9 Hz, Ar-Dmab), 5.09 (2H, s, Ar-CH2O-Dmab), 4.40–4.14
141.3 (3C, CAr-OPfp, C4a-, C4b-Fmoc), 140.2, 138.9 (1C each, CAr
-
OPfp), 136.4 (2C, CAr-OPfp), 136.1 (1C, CAr-CH2O-Dmab), 129.2
(2C, CAr-Dmab) 127.6 (2C, C3-, C6-Fmoc), 127.0 (2C, C2-, C7-Fmoc),
126.6 (2C, CAr-Dmab), 125.1 (2C, C1-, C8-Fmoc), 119.8 (2C, C4-, C5-
Fmoc), 107.4 (1C, C@CN), 66.8 (1C, CH2-Fmoc), 65.9 (1C, CAr-CH2O-
Dmab), 54.1 (1C, C
a
-Met), 52.4 (2C, 2 ꢂ CH2 ring-Dmab), 48.8 (1C,
Ca-Asp), 47.2 (1C, C9-Fmoc), 39.2 (1C, CH2CH-Dmab), 35.4 (1C, Cb-
Asp), 31.4 (1C, b-CH2-Met), 29.8 (1C, Cd-Met), 29.5 (1C, CH2CH-
Dmab), 27.2 (2C, 2 ꢂ CH3 ring-Dmab), 21.7 (2C, CH2CH(CH3)2-
Dmab), 14.1 (1C, SCH3-Met). MALDI-TOFMS: m/z 986.1 (M+Na),
963.6 (M+H).
3.1.20. Glycopeptide 25
To a stirred mixture of the glycopeptide 22 (75 mg, 65
lmol)
and octanethiol (0.11 mL, 0.65 mmol), DBU (5 L, 33 mol) was
l
l
added, and the reaction mixture was stirred for 1 h. The solvent
was evaporated to dryness, and the residue was washed several
times with warm Et2O. The amine product 23 was not purified fur-
ther but was used directly in the coupling reaction with the pro-
tected dipeptide 24.

1426
B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
To a mixture of the amine 23 (20.6 mg, 22
Met-Asp(ODmab)-Opfp (24) (21 mg, 22 mol) in dry THF, HOBt
(5 mg, 33 mol) was added, and the reaction mixture was stirred
l
mol) and Fmoc-
a/b mixture of the aspartimide side product (6.4 mg, 88%) as a
l
white solid, after lyophilization in dioxane. 1H NMR (500 MHz,
MeOD) of the major isomer: d 7.60 (1H, d, J = 7.8 Hz, H4-Trp),
7.32 (1H, d, J = 8.8 Hz, H7-Trp), 7.11–7.05 (2H, m, H2-, H6-Trp),
7.01 (1H, br t, J = 7.1 Hz, H5-Trp), 5.17 (2H, br s, H10-rha), 5.11
l
at rt for 3 h. The solvent was then removed under vacuum, and
the residue was purified by flash chromatography (2:1 hexanes–
EtOAc) to afford the protected glycopeptide 25 as a white, crystal-
line solid containing a minor peptide impurity. Further purifica-
tion by preparative HPLC (C-18 reversed phase, 80:20 to 5:95
H2O–CH3CN over 30 min) gave the pure glycopeptide 25 as a col-
orless solid (28 mg, 72%). 1H NMR (500 MHz, CD2Cl2): d 8.45 (1H,
br s, NH-Trp ring), 7.82–7.76 (2H, m, H4-, H5-Fmoc), 7.65–7.55
(3H, m, H1-, H8-Fmoc, H7-Trp), 7.45–7.38 (3H, m, ArH-Dmab,
H3-, H6-Fmoc), 7.38–7.30 (4H, m, H2-, H7-Fmoc, H4-Trp), 7.29–
7.26 (1H, br d, J = 8.7 Hz, NH-Asp), 7.19–7.12 (2H, m, H5-Trp,
ArH-Dmab), 7.12–7.07 (2H, m, H6-Trp, ArH-Dmab) 7.04 (1H, br
s, H2-Trp), 6.77 (1H, br d, J = 7.8 Hz, NH-Trp), 5.34 (1H, br s,
NH-Dmab), 5.32–5.24 (3H, m, H2-, H2”, H300-rha), 5.23 (1H, s,
H1-rha), 5.17–4.97 (6H, m, H30-, H4-, H40-, H4”-rha, Ar-CH2O-
Dmab), 4.94 (1H, d, J = 1.50 Hz, H10-rha), 4.79 (1H, br s, H1”-
(1H, br s, H1-rha), 4.91 (1H, br s, H1”-rha), 4.40–4.31 (1H, m,
H-Trp), 4.26 (1H, dd, J = 4.9 Hz, -H-Asp), 4.14 (1H, dd,
J = 6.4 Hz,
-H-Met), 4.02 (1H, br s, H2-rha), 3.97 (2H, br s, H20-,
a-
a
a
H2”-rha), 3.93 (1H, m, H5-rha), 3.86 (1H, dd, J = 9.5 and 3.2 Hz,
H30-rha), 3.75–3.63 (4H, m, H50-, H5”, H3, H3”-rha), 3.52 (1H,
dd, J = 9.7, H4-rha), 3.40–3.31 (2H, m, H40-, H4”-rha), 3.09–2.67
(2H, m, b-CH2-Trp), 2.86–2.72 (3H, m, b-CH2-Asp, CH2S-Trp),
2.68–2.52 (3H, m, b-CH2-Asp, dCH2-Met), 2.19–2.10 (2H, m,
bCH2-Met), 2.09 (3H, s, SCH3-Met), 1.32–1.20 (9H, m, 3 ꢂ CH3-
rha). 13C NMR (125 MHz, MeOD) of the major isomer: d 177.4,
174.2, (3C, 3 ꢂ C@O), 136.5, 127.8, 124.7, 123.1, 121.0, 118.6,
118.2, 111.1 (each, 1C, 8 ꢂ CAr-Trp), 102.7 (1C, C100-rha), 101.3
(1C, C10-rha), 86.6 (1C C1-rha,), 78.2 (1C, C20-rha), 73.2, 72.9,
72.5, 72.3 (each 1C, 3 ꢂ C4-, C2-rha), 71.1, 70.9, 70.7, 70.6, 70.1,
69.1, 68.9, (10C, C2, 3 ꢂ C3-, 3 ꢂ C4-rha, 3 ꢂ C5-rha), 53.4, 52.3
rha), 4.77–4.72 (1H, m,
6.4 Hz, CH2-Fmoc), 4.43–4.32 (2H, m, CH2-Fmoc,
(1H, dd, J = 7.32 and 6.4 Hz, H9-Fmoc), 4.18–4.07 (2H, m, H5-
rha, -H-Met), 4.04 (1H, dd, J = 9.28 and 3.42 Hz, H3-rha), 3.97
aH-Fsp), 4.47 (1H, dd, J = 10.7 and
a-H-Trp), 4.24
51.7 (each 1C, Ca-Met, Ca-Asp, Ca-Trp), 50.1 (1C, b-CH2-Asp),
35.5 (1C, SCH2-Trp), 30.9 (1C, b-CH2-Met), 29.6 (1C, b-CH2-Trp),
28.3 (1C, SCH2-Met), 16.9, ((1C, C6-rha), 16.7 (2C, 2 ꢂ C6-rha),
13.9 (1C, SCH3-Met). HRMS: Calcd for [C38H56N4O15S2 + Na]: m/z
895.3076. Found: m/z 895.3075.
a
(1H, m, H5”-rha), 3.91 (1H, br s, H20-rha), 3.84 (1H, m, H50-rha),
3.07–2.89 (4H, m, b-CH2-Trp, CH2CH-Dmab, b-CH2-Asp), 2.88–
2.72 (3H, m, b-CH2-Asp, CH2S), 2.51–2.49 (2H, m, d-CH2-Met),
2.48 (2H, s, CH2 ring-Dmab), 2.36 (2H, s, CH2 ring-Dmab), 2.19–
1.98 (26H, m, SCH3-Met, b-CH2-Met, 7 ꢂ OAc), 1.87–1.77 (1H, m,
CH2CH-Dmab), 1.21–1.13 (9H, m, 3 ꢂ CH3-rha), 1.07 (6H, s,
2 ꢂ CH3 ring-Dmab), 0.71 (6H, d, J = 6.8 Hz, CH2CH(CH3)2-Dmab).
3.1.22. Glycopeptide 1
The amine product 29 (11 mg, 7.3 lmol) was dissolved in 2%
NH2NH2 in THF (5 mL), and the mixture was stirred for 2 h at rt.
The solvent was evaporated, and the residue 30 was dissolved in
MeOH (5 mL) and MeONa–MeOH (five drops) was added. The reac-
tion mixture was stirred overnight at rt. Rexin H+ 101 was then
added until the pH was slightly basic, the resin was filtered, and
the filtrate was further neutralized with HOAc. The solvent was
evaporated, and the crude product was washed successively with
hexanes (10 mL), CH2Cl2 (10 mL), and EtOAc (10 mL). The crude
material was further purified by HPLC (C-18 reversed phase,
95:5–5:95 H2O–CH3CN over 15 min) to afford the desired glyco-
peptide 1 (6.5 mg, 68%) as a white solid. 1H NMR (see above):
HRMS: Calcd for C38H58N4O16S2: m/z 891.3367. Found: m/z
891.3356.
13C NMR (150 MHz, CD2Cl2):
d 176.3 (2C, C@O ring-Dmab),
171.8–169.5 (10C, 11 ꢂ C@O), 156.7 (1C, C@O-urethane), 144.0
(2C, C8a-, C9a-Fmoc), 143.9 (1C, CAr-N-Dmab), 141.5 (2C, C4a-,
C4b-Fmoc), 137.3 (1C, C3a-Trp), 136.4 (1C, CAr-CH2O- Dmab),
129.1 (2C, CAr-Dmab), 128.0 (1C, C7a-Trp), 127.9 (3C, C3-, C6-
Fmoc), 127.3 (2C, C2-, C7-Fmoc), 126.9 (2C, CAr-Dmab), 125.2
(2C, C1-, C8-Fmoc), 123.3 (1 C, C2-Trp), 122.2 (1 C, C6-Trp),
120.2 (2 C, C4-, C5-Fmoc), 119.6 (1 C, C5-Trp), 118.9 (1C, C7-
Trp), 111.4 (1C, C4-Trp), 111.4 (1C, C3-Trp), 100.2 (1C, C1”-rha),
99.8 (1C, C10-rha), 83.2 (1C, C1-rha), 77.6 (1C, C20-rha), 75.2 (1C,
C3-rha), 73.1 (1C, C2-rha), 72.9 (1C, C4”-rha), 70.9 (1C, C30-rha),
70.3 (1C, C4-rha), 70.3 (1C, C40-rha), 69.9 (1C, C2”-rha), 68.9 (1C,
C3”-rha), 67.6 (1C, C50-rha), 67.4 (1C, C5-rha), 67.3 (1C, C5”-rha),
3.2. Molecular modeling
67.2 (1C, CH2-Fmoc), 66.0 (1C, CAr-CH2O-Dmab), 54.9 (1C, C
Met), 53.9 (1C, CH2 ring-Dmab), 52.4 (1C, CH2 ring-Dmab), 50.4
(1C, C -Trp), 49.8 (1C, C -Asp), 47.4 (1C, C9-Fmoc), 38.2 (1C,
a-
The molecular structures of the glycopeptides 1 and 2 were con-
structed using SYBYL 6.6 (Tripos, Inc.) with the coordinates of the
two 1.8 Å resolution crystal structures of the Fab fragment of
SYA/J6 complexed with the octapeptide and the pentasaccharide
(PDB entry 1PZ5).22 The structures of 1 and 2 were then optimized
by energy minimization within the binding site to achieve a
reasonable structure in each case, employing the standard
Tripos molecular mechanics force field49 and Gasteiger–Marsili
charges,50,51 with a 0.001 kcal/mol energy gradient convergence
criterion. The atom charges were retained on both 1 and 2 for
the docking calculations. The water was removed. Only polar
hydrogens were added to the protein, and Kollman united-atom
partial charges52 were assigned. Compounds 1 and 2 were then
docked into the antibody-combining site of the Fab fragment of
SYA/J6 using AUTODOCK 3.0.26 All active torsions of 1 and 2 were se-
lected to be fully flexible during the docking experiments. The grid
maps were constructed using 70 ꢂ 70 ꢂ 70 points, with grid point
spacing of 0.375 Å. A Lamarckian Genetic Algorithm (LGA) was
used to search each conformation space for low-energy binding
orientations. The default setting was adopted, and 500 LGA docking
runs were performed.
a
a
CH2CH-Dmab), 35.7 (1C, Cb-Asp), 31.7 (1C, b-CH2-Met), 29.9 (1C,
Cd-Met), 29.6 (1C, -CH2CH-Dmab), 29.3 (1C, b-CH2-Trp), 28.1
(2C, 2 ꢂ CH3 ring-Dmab), 22.5 (2C, CH2CH(CH3)2-Dmab), 21.1–
20.7 (7C, 7 ꢂ CH3-OAc), 17.5–17.1 (3C, 3 ꢂ CH3-rha), 15.3 (1C,
SCH3-Met), MALDI-TOFMS: m/z 1741.7 (M+Na), 1757.1 (M+K),
1718.6 (M+H). Anal. Calcd for C87H107N5O27S2: C, 60.79; H, 6.27;
N, 4.07. Found: C, 60.66; H, 6.45; N, 3.71.
3.1.21. Aspartimide 36
To a stirred mixture of 25 (13 mg, 7.6
lmol) and octanethiol
(13 L, 76 mol), DBU (0.6 L, 33 mol) was added, and the reac-
l
l
l
l
tion mixture was stirred for 1 h. The solvent was evaporated to
dryness, and the residue was washed several times with warm
Et2O. The amine product 29 was then dissolved in MeOH (3 mL)
and MeONa–MeOH (five drops) was added. The reaction mixture
was stirred overnight at rt. Rexin H+ 101 was then added until
the pH was neutral. The resin was removed by filtration, the sol-
vent was concentrated, and the crude product was purified by
flash chromatography (10:3:1 EtOAc–MeOH–H2O) to afford a 2:1

B. R. Hossany et al. / Carbohydrate Research 344 (2009) 1412–1427
1427
12. Barington, T.; Gyhrs, A.; Kristensen, K.; Heilmann, C. Infect. Immun. 1994, 62,
9–14.
3.3. Immunochemical analysis
13. Peeters, C.; Tenbergenmeekes, A. M.; Poolman, J. T.; Beurret, M.; Zegers, B. J. M.;
Rijkers, G. T. Infect. Immun. 1991, 59, 3504–3510.
14. Johnson, M. A.; Pinto, B. M. Top. Curr. Chem. 2008, 273, 55–116. and references
cited therein.
15. Johnson, M. A.; Pinto, B. M. Aust. J. Chem. 2002, 55, 13–25.
16. Borrelli, S.; Hossany, R. B.; Pinto, B. M. Clin. Vaccine Immunol. 2008, 15, 1106–
1117.
17. Borrelli, S.; Johnson, M. A.; Hossany, R. B.; Findlay, S. ACS Symp. Ser. 2007, 989,
335.
3.3.1. Lipopolysaccharide, and O-polysaccharide from S. flexneri
Y, peptide MDWNMHAA, and monoclonal antibody SYA/J6, used
in ELISA
The lipopolysaccharide (LPS) and O-polysaccharide from
S. flexneri Y and monoclonal antibody SYA/J6 (IgG3) were provided
by Dr. D. R. Bundle21,22,31,44–48 and were used as antigens/inhibi-
tors. The mimetic-peptide MDWNMHAA,24 was used as an inhibi-
tor in ELISA.
18. Harris, S. L.; Craig, L.; Mehroke, J. S.; Rashed, M.; Zwick, M. B.; Kenar, K.; Toone,
E. J.; Greenspan, N.; Auzanneau, F. I.; MarinoAlbernas, J. R.; Pinto, B. M.; Scott, J.
K. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2454–2459.
19. Borrelli, S.; Hossany, R. B.; Findlay, S.; Pinto, B. M. Am. J. Immunol. 2006, 2, 73–83.
20. Hale, T. L. In Topley and Wilson’s Microbiology and Microbial Infections; Hansler,
W. J., Shumman, M., Eds.; Arnold: London, 1998; pp 479–493.
21. Vyas, N. K.; Vyas, M. N.; Chervenak, M. C.; Johnson, M. A.; Pinto, B. M.; Bundle,
D. R.; Quiocho, F. A. Biochemistry 2002, 41, 13575–13586.
22. Vyas, N. K.; Vyas, M. N.; Chervenak, M. C.; Bundle, D. R.; Pinto, B. M.; Quiocho, F.
A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 15023–15028.
23. Beenhouwer, D. O.; May, R. J.; Valadon, P.; Scharff, M. D. J. Immunol. 2002, 169,
6992–6999.
24. Hossany, R. B.; Johnson, M. A.; Eniade, A. A.; Pinto, B. M. Bioorg. Med. Chem.
2004, 12, 3743–3754.
25. Horton, D.; Wander, J. D. In The Carbohydrates; Pigman, W. W., Horton, D., Eds.;
Academic: New York, 1980; Vol. 1A, p 799.
26. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639–1662.
27. Auzanneau, F. I.; Forooghian, F.; Pinto, B. M. Carbohydr. Res. 1996, 291, 21–41.
28. Hoog, C.; Rotondo, A.; Johnston, B. D.; Pinto, B. M. Carbohydr. Res. 2002, 337,
2023–2036.
3.3.2. Competitive-inhibition ELISA studies with
lipopolysaccharide and O-polysaccharide of S. flexneri Y, peptide
MDWNMHAA, and glycopeptide as inhibitors
Competitive–inhibition ELISA was performed in 96-well plates
(NUNC-MaxiSorp, Rochester, NY), coated overnight with 100
lL/
well of LPS from S. flexneri Y (10 g/mL in carbonate buffer pH
l
9.6). Wells were blocked by the addition of 1% BSA for 2 h at room
temperature. The plates were washed three times with a washing
solution of 0.05% Tween 20 and 0.9% NaCl. Monoclonal antibody
SYA/J6 (ꢁ1
serial dilutions of inhibitors in PBS-T, starting at an initial concen-
tration of 400 g/mL, for 1 h at room temperature. Then, 100 L of
lg/mL) was pre-incubated in duplicate with twofold
l
l
the mixtures was transferred to plates, and the plates were incu-
bated for 3 h at room temperature. After washing four times as be-
29. Pozgay, V. Carbohyd. Res. 1992, 235, 295–302.
fore, 100 lL per well of alkaline phosphatase-labeled goat anti-
30. Kartha, K. P. R.; Field, R. A. Tetrahedron Lett. 1997, 38, 8233–8236.
31. Hecht, S. Recognition of Carbohydrate Antigens by Antibody Binding Sites. In
The Carbohydrates; Oxford University: Oxford, 1998; pp 370–440.
32. Zhu, X. M.; Schmidt, R. R. Chem. Eur. J. 2004, 10, 875–887.
33. Mergler, M.; Dick, F. J. Pept. Sci. 2005, 11, 650–657.
34. Hvidt, T.; Szarek, W. A.; Maclean, D. B. Can. J. Chem. 1988, 66, 779–782.
35. Sheppeck, J. E.; Kar, H.; Hong, H. Tetrahedron Lett. 2000, 41, 5329–5333.
36. Cammish, L. E.; Kates, S. A. In Fmoc Solid Phase Peptide Synthesis: A Practical
Approach; Chan, W. C., White, P. D., Eds.; Oxford University: Oxford, 2000; p
277.
37. Dourtoglou, V.; Gross, B.; Lambropoulou, V.; Zioudrou, C. Synthesis 1984, 7,
572–574.
38. Dourtoglou, V.; Ziegler, J. C.; Gross, B. Tetrahedron Lett. 1978, 15, 1269–1272.
39. Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahedron Lett. 1989, 30,
1927–1930.
mouse IgG (Caltag Laboratories, San Francisco, CA) diluted
1:3000 in PBS-T was added. The plates were incubated overnight
at room temperature, and were again washed four times. Substrate
solution (100 lL) containing p-nitrophenyl phosphate (1 mg/mL,
Kirkegaard & Perry lab, Gaithersburg, MD) was added to the wells.
After 25–30 min at room temperature, the plates were scanned at
405 nm in a SpectraMax 340 microplate reader. The percentage of
inhibition was determined by the decrease in A405 value in wells
with inhibitors as compared with those without inhibitors.
Acknowledgments
40. Chan, W. C.; Bycroft, B. W.; Evans, D. J.; White, P. D. J. Chem. Soc., Chem.
Commun. 1995, 2209–2210.
41. Barlos, K.; Gatos, D. In Fmoc Solid Phase Peptide Synthesis: A Practical Approach;
Chan, W. C., White, P. D., Eds.; Oxford University: Oxford, 2000; pp 215–228.
42. Purification of Laboratory Chemicals; Perrin, D. D., Armarego, W. L. F., Eds., 3rd
ed.; Pergamon: London, 1988.
43. Kaiser, E.; Colescot, R.; Bossinge, C.; Cook, P. I. Anal. Biochem. 1970, 34, 595–598.
44. Altman, E.; Bundle, D. R. Methods Enzymol. 1994, 247, 243–253.
45. Bundle, D. R.; Gidney, M. A.; Josephson, S.; Wessel, H.-P. ACS Symp. Ser. 1983,
231, 49–63.
We are grateful to D. R. Bundle for the generous gifts of the SYA/
J6 antibody, the LPS, and O-polysaccharide, and to the Natural Sci-
ences and Engineering Research Council of Canada for financial
support.
References
46. Bundle, D. R. Pure Appl. Chem. 1989, 61, 1171–1180.
47. Bundle, D. R.; Altman, E.; Auzanneau, F. I.; Baumann, H.; Eichler, E.; Sigurskjold,
B. W.. In Alfred Benzon Symp. Ser; Bock, K., Clausen, H., Eds.; Munksgaard:
Copenhagen, 1994; vol. 36, pp 168–181.
48. Vyas, M. N.; Vyas, N. K.; Meikle, P. J.; Sinnott, B.; Pinto, B. M.; Bundle, D. R.;
Quiocho, F. A. J. Mol. Biol. 1993, 231, 133–136.
1. Grandi, G. Trends Biotechnol. 2001, 19, 181–188.
2. Jennings, H. J. Curr. Top. Microbiol. Immunol. 1990, 150, 97–127.
3. Jennings, H. J. Adv. Exp. Med. Biol. 1988, 228, 495–550.
4. Lee, C. J. Mol. Immunol. 1987, 24, 1005–1019.
5. Lindberg, A. A. Vaccine 1999, 17, S28–36.
6. Weintraub, A. Carbohydr. Res. 2003, 338, 2539–2547.
7. Mc Cool, T. L.; Harding, C. V.; Greenspan, N. S.; Schreiber, J. R. Infect. Immun.
1999, 67, 4862.
8. Roy, R. Drug Discovery Today: Technol. 2004, 1, 327–336.
9. Mond, J. J.; Lees, A.; Snapper, C. M. Annu. Rev. Immunol. 1995, 13, 655–692.
10. Parker, D. C. Annu. Rev. Immunol. 1993, 11, 331–360.
11. Gonzalez-Fernandez, A.; Faro, J.; Fernandez, C. Vaccine 2008, 26, 292–300.
49. Clark, M.; Cramer, R. D.; Vanopdenbosch, N. J. Comput. Chem. 1989, 10, 982–
1012.
50. Gasteiger, J.; Marsili, M. Tetrahedron 1980, 36, 3219–3228.
51. Gasteiger, J.; Marsili, M. Org. Magn. Reson. 1981, 15, 353–360.
52. Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M.; Ferguson, D. M.;
Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A. J. Am. Chem. Soc. 1995,
117, 5179–5197.